CN116529378A

CN116529378A - Plant regulatory element and use thereof for automatic excision

Info

Publication number: CN116529378A
Application number: CN202180071059.8A
Authority: CN
Inventors: P·J·托; Z·瓦赫希帕瓦拉; 叶旭东
Original assignee: Monsanto Technology LLC
Current assignee: Monsanto Technology LLC
Priority date: 2020-10-20
Filing date: 2021-10-19
Publication date: 2023-08-01
Also published as: WO2022086951A1; CA3196238A1; EP4232587A1; US20240035039A1

Abstract

Recombinant DNA molecules and constructs for modulating gene expression in plants are provided. By expressing the recombinase encoded by the recombinant DNA molecule or construct, one or more expression cassettes of the recombinant DNA molecule or construct can be excised from the transformed transgenic plant by means of the presence of flanking site-specific recombination sites in the recombinant DNA molecule or construct. Such expression cassettes can be removed from plants transformed with the recombinant DNA constructs or vectors using such recombinase systems. The recombinase transgene may be operably linked to a tissue-preferred or tissue-specific promoter for automatic excision in the transformed plant without crossing with a different transgenic line expressing the recombinase. Also provided are methods of causing automatic excision of one or more expression cassettes in transgenic plants and cells containing or transformed with the recombinant DNA molecules or constructs of the disclosure.

Description

Plant regulatory element and use thereof for automatic excision

Citation of related application

The present application claims the benefit of U.S. provisional application No. 63/093,893, filed on even date 10/20 in 2020, which is incorporated herein by reference in its entirety.

Incorporation of the sequence Listing

The sequence listing contained in the file named "MONS512WO_ST25.Txt" is 111,683 bytes (as in Microsoft @Measured in (a), created at 10.18 of 2021, which is submitted together in an electronic submission and incorporated herein by reference.

Technical Field

The present invention relates to the fields of plant molecular biology and plant genetic engineering. More specifically, the present invention relates to DNA molecules for modulating the expression of a site-specific recombinase gene in plants.

Background

Regulatory elements are genetic elements that regulate gene activity by regulating transcription of an operably linked transcribable DNA sequence. Such elements may include promoters, leader sequences, introns and 3' untranslated regions, and may be used in the fields of plant molecular biology and plant genetic engineering.

The use of transgenic technology provides many beneficial traits for agricultural purposes, but also encounters some challenges. One concern relates to the presence of marker genes conferring antibiotic or herbicide resistance in transgenic crop plants. In addition, there may be other transgene cassettes or DNA sequences that are designed for specific purposes and are present in the initial transformation, but are not required in the final transgene product. In the field of plant biotechnology, it is highly desirable to remove such marker genes, as well as other unwanted expression cassettes and DNA sequences.

Many strategies have been devised for generating marker-free transgenic plants. For example, removal of the marker gene expression cassette may be performed using a double T-DNA transformation system or a site-specific recombinase system.

Double T-DNA transformation systems utilize two comprising two independent T-DNAsMeta-plant transformation vectors (double T-DNA transformation system). One T-DNA comprises a marker gene expression cassette. The other T-DNA comprises an expression cassette for the gene of interest that is expected to remain in the transgenic plant. Plant cell transformation may be performed by agrobacterium-mediated transformation. Each T-DNA may be integrated into a separate chromosome of the genome of the transformed plant cell. After transformation and plant regeneration, R is allowed to stand ₀ Plant selfing to produce R ₁ And (5) offspring. Selecting R having T-DNA comprising an expression cassette intended for the final transgene product but lacking T-DNA comprising a marker gene expression cassette ₁ Progeny plants (see, e.g., komari, T.et al, (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection mark ers, the Plant Journal,10 (1): 165-174). The double T-DNA transformation system has some drawbacks in terms of efficiency. In the double T-DNA transformation system, transformant R ₀ Plants may have more than one copy of one or two T-DNA, which may have to be excluded, and the percentage of plants selected with only one copy of each T-DNA may be low.

Another system for removing marker gene expression cassettes from transgenic plants relies on excision by use of a site-specific recombinase. A number of Site-specific recombinases may be used, such as Cre-recombinase, flp-recombinase (Lyznik, L. Et al, (2000) Gene Transfer Mediat ed by Site-Specific Recombination Systems, plant Molecular Biology Manual N1, 1-26), R-recombinase (Machida, C. Et al, (2000) Use of the R-RS Site-Specific Recombination System in Plants, plant Molecular Biol ogy Manual N2, 1-23) or Gin-recombinase (Maeser, S. Et al, (1991) Gin recombinase of phage Mu may catalyze Site-specific recombination in plant protoplasts, mol Gen Genet, 230:170-176). Essentially, in constructs such as T-DNA insertions, marker gene expression cassettes are flanked by site-specific recombinase recognition sequences such that the construct sequence between the site-specific recombinase recognition sequences can be excised by expression of the recombinase. The expression cassette that remains in the transgenic plant after excision is expected to be present in the construct outside the site-specific recombinase recognition sequence of the construct.

Removal of the expression cassette flanked by site-specific recombinase recognition sequences can be accomplished using hybridization strategies or by automated excision. In a crossing strategy, a plant (e.g., R1 progeny) that is preferably homozygous for the presence of the construct is crossed with another transgenic plant line transformed with an expression cassette for expression of the site-specific recombinase. The resulting F is then selected for the presence of the construct ₁ The progeny, the construct has had the expression cassette flanked by site-specific recombinase recognition sequences excised. In the case of automatic excision, another expression cassette encoding a site-specific recombinase is present in the construct, wherein the other expression cassette will be excised either between or on both sides of the site-specific recombinase recognition sequence, such that all such expression cassettes are excised by the site-specific recombinase. Promoters typically have a preference or specificity for driving expression in a particular type of cell or tissue. Not all promoters and expression elements are suitable for efficient automatic excision, and extensive experimentation is required to identify the correct promoter to drive expression of the recombinase and additional expression elements to regulate expression of the recombinase, such as introns and 3' UTRs, to achieve the desired excision frequency and result.

There is a need to drive expression elements in crop plants that are effectively self-resected. The present disclosure provides several expression elements identified by years of experimentation that can be used to drive expression of a recombinase and produce efficient automatic excision of markers and/or recombinase transgenes and possibly other expression cassettes in many crop species after transformation.

Disclosure of Invention

The present invention provides gene regulatory elements for driving site-specific recombinases in plants that will result in efficient automatic excision of marker gene expression cassettes as well as expression cassettes for genome editing. The invention also provides recombinant DNA molecule constructs comprising the regulatory elements. The invention also provides constructs comprising the regulatory elements. In one embodiment, the regulatory element is operably linked to the site-specific recombinase. In certain embodiments, the regulatory element is a meiosis promoter. In other embodiments, the regulatory element is comprised in a construct comprising at least three transgene cassettes. The invention also provides methods of using the regulatory elements and of making and using recombinant DNA molecules and constructs comprising the regulatory elements.

Thus, in one aspect, the present invention provides a recombinant DNA molecule comprising a DNA regulatory sequence selected from the group consisting of: (a) A sequence having at least about 80% sequence identity to any one of SEQ ID NOs 1-26, 59-62 and 64-66; (b) A sequence comprising any one of SEQ ID NOs 1-26, 59-62 and 64-66; and (c) (i) any of SEQ ID NOS: 1-26, 59-62 and 64-66 or (ii) a fragment of any sequence having at least 80% sequence identity to any of SEQ ID NOS: 1-26, 59-62 and 64-66, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable DNA sequence encoding a site-specific recombinase. In particular embodiments, the recombinant DNA molecule comprises a DNA regulatory sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the DNA sequence of any one of SEQ ID NOs 1-26, 59-62 and 64-66. In particular embodiments, the DNA regulatory sequences include regulatory elements having gene regulatory activity. In some embodiments, the regulatory element comprises a promoter. In still other embodiments, the regulatory element comprises an intron. In still other embodiments, the regulatory element comprises a 3' utr. In still other embodiments, the DNA regulatory sequence is a germline-preferred promoter. In other embodiments, the germline preference promoter is selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 10, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 17, SEQ ID NO. 21 and SEQ ID NO. 65. In other embodiments, the germline-preferred promoter is the CDC45 promoter. In yet a further embodiment, the CDC45 promoter is selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 5 and SEQ ID NO. 10, and sequences having at least 80% sequence identity to any of SEQ ID NO. 2, 5, 7, 10, 13, 14, 17, 21 and 65. In still other embodiments, the DNA regulatory sequence is an embryo-preferred promoter. In other embodiments, the DNA regulatory sequence is SEQ ID NO. 60 or a sequence having at least 80% sequence identity to SEQ ID NO. 60. In still other embodiments, the heterologous transcribable DNA sequence comprises a gene encoding a site-specific recombinase. In other embodiments, the site-specific recombinase is selected from the group consisting of: cre-recombinase, flp-recombinase, R-recombinase and Gin-recombinase. In yet another embodiment, the site-specific recombinase is Cre-recombinase.

In another aspect, the recombinant DNA construct further comprises one or both of the following expression cassettes: a selectable marker transgene; and/or transgenes of agronomic interest. In another embodiment, the recombinant DNA construct further comprises a pair of site-specific recombination site sequences flanking one or both transcribable DNA sequences encoding a site-specific recombinase and/or a selectable marker transgene, wherein the site-specific recombination site is cleavable by the site-specific recombinase. In a further embodiment, the selectable marker transgene of the recombinant DNA construct confers resistance to a herbicide or antibiotic. In other embodiments, the site-specific recombination site sequences of the recombinant DNA constructs are each selected from the group consisting of: loxP, lox.TATA-R9, FRT, RS and GIX. In a specific embodiment, the site-specific recombination site sequences of the recombinant DNA constructs are each LoxP or Lox. TATA-R9 sites. In other embodiments, the site-specific recombination site sequences of the recombinant DNA constructs each comprise SEQ ID NO. 44 or SEQ ID NO. 45.

In another aspect, the agronomically beneficial transgene of the recombinant DNA construct confers herbicide tolerance in the plant. In some embodiments, the agronomically beneficial transgene of the recombinant DNA construct confers insect resistance or disease resistance in plants. In further embodiments, agronomically beneficial transgenes of the recombinant DNA constructs confer increased yield or stress tolerance in plants. In still other embodiments, the agronomically beneficial transgene of the recombinant DNA construct encodes a dsRNA, miRNA, or siRNA.

In another aspect, the recombinant DNA construct further comprises one or both of: an expression cassette encoding a guide RNA; and/or an expression cassette encoding a site-specific nuclease. The recombinant DNA construct further comprises a site-specific recombination site sequence flanking the one or more transcribable DNA sequences encoding the site-specific recombinase, a selectable marker transgene, an expression cassette encoding the guide RNA, and/or an expression cassette encoding the site-specific nuclease, wherein the site-specific recombination site is cleavable by the site-specific recombinase. In further embodiments, the guide RNA comprises a targeting sequence that targets a sequence in the genome of the eukaryotic cell for genome editing or site-specific integration. In another embodiment, the eukaryotic cell is a plant cell. In yet another embodiment, the recombinant DNA construct comprises two or more expression cassettes encoding two or more guide RNAs. In a further embodiment, the recombinant DNA construct comprises two, three, four, five, six, seven, eight, nine or ten different expression cassettes encoding guide RNAs. In further embodiments, the site-specific nuclease is an RNA-guided endonuclease or a CRISPR-associated nuclease. In another embodiment, the RNA guided endonuclease or CRISPR-associated nuclease is selected from the group consisting of: cas1, cas1B, cas2, cas3, cas4, cas5, cas6, cas7, cas8, cas9, cas10, cpf1, cys2, csy3, cse1, cse2, csc1, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmr1, cmr3, cmr4, cmr5, cmr6, csb1, csb2, csb3, csx17, csx14, csx10, csx16, csaX, csx3, csx1, csx15, csf1, csf2, csf3, csf4, casX and CasY. In specific embodiments, the RNA guided endonuclease or CRISPR-associated nuclease is Cpf1 or Cas9.

In another aspect, provided herein are DNA molecules or vectors comprising recombinant DNA constructs. In another embodiment, the DNA transformation vector comprises a recombinant DNA construct and a T-DNA segment bounded by a left border and a right border. In a further embodiment, the transcribable DNA sequence encoding the site-specific recombinase is located between the left and right boundaries of the T-DNA segment within the DNA transfer vector. In yet another embodiment, the DNA transformation vector comprises a recombinant DNA construct and a T-DNA segment having a left border and a right border, wherein one or more of a transcribable DNA sequence encoding a site-specific recombinase, a selectable marker transgene, and/or an agronomically beneficial transgene is located between the left border and the right border of the T-DNA segment. In further embodiments, the DNA transformation vector comprises a recombinant DNA construct and a T-DNA segment having a left border and a right border, wherein one or more of a transcribable DNA sequence encoding a site-specific recombinase, a selectable marker transgene, an agronomically beneficial transgene, an expression cassette encoding a guide RNA, and/or an expression cassette encoding a site-specific nuclease is located between the left border and the right border of the T-DNA segment.

In another aspect, provided herein are transgenic plants, plant parts, or plant cells comprising the recombinant DNA constructs. The recombinant DNA construct is stably transformed into the genome of a transgenic plant, plant part or plant cell. The transgenic plant, plant part or plant cell is a maize, soybean, cotton or canola plant, plant part or plant cell. Also provided herein are bacterial cells comprising the recombinant DNA constructs or transformation vectors.

In another aspect, provided herein is a method for producing a transgenic plant or plant part, comprising (a) transforming a plant cell of an explant with a DNA molecule or vector comprising a recombinant DNA construct to produce one or more transformed plant cells comprising the recombinant DNA construct stably transformed into the genome of the one or more transformed plant cells; (b) Regenerating or producing a transgenic plant from an explant, wherein the transgenic plant comprises a recombinant DNA construct stably transformed into the genome of one or more cells of the transgenic plant. In one embodiment, the plant cells are transformed via agrobacterium-mediated transformation or rhizobia-mediated transformation. In another embodiment, plant cells are transformed via particle-mediated transformation or particle bombardment-mediated transformation. In yet another embodiment, the transgenic plant and plant cell are maize, soybean, cotton or canola plants and plant cells, respectively. In yet another embodiment, the method further comprises: (c) Isolating or harvesting a plant part from said transgenic plant.

In another aspect, provided herein is a method of excision of an expression cassette from the genome of a transgenic plant, the method comprising: (a) Transforming a plant cell with a DNA molecule or vector comprising the recombinant DNA construct of any one of claims 13-30 to produce one or more transformed plant cells comprising the recombinant DNA construct stably transformed into the genome of the one or more transformed plant cells; (b) Regenerating or producing a transgenic plant at least in part from the one or more stably transformed plant cells; (c) Crossing the transgenic plant with itself or another plant; and (d) selecting one or more progeny plants in which one or both of the transcribable DNA sequences encoding the site-specific recombinase and/or the selectable marker transgene between the pair of site-specific recombination site sequences of the recombinant DNA construct is excised and no longer present in the genome of the progeny plant. In a further embodiment of the method, the recombinant DNA construct further comprises one or both of the following expression cassettes located between the pair of site-specific recombination site sequences of the recombinant DNA construct: and wherein one or more progeny plants are selected in which one or more of the transcribable DNA sequence encoding the site-specific recombinase, the selectable marker transgene, the expression cassette encoding the guide RNA, and/or the expression cassette encoding the site-specific nuclease of the recombinant DNA construct is excised and no longer present in the genome of the progeny plant. In particular embodiments, the transgenic plant and plant cell are maize, soybean, cotton or canola plants and plant cells, respectively. In another embodiment, the method further comprises (e) isolating or harvesting plant parts from one or more of the progeny plants. In yet another embodiment, the method further comprises (f) crossing one or more of the progeny plants with itself or another plant.

Brief description of the sequence

SEQ ID NO. 1 is a DNA sequence of the regulatory expression element group (EXP) EXP-Zm.Cdc45-1+Zm.DnaK:1:1 comprising a promoter (P-Zm.Cdc45-1:8) operably linked to 5 'of a leader sequence (L-Zm.Cdc45-1:1) operably linked to 5' of an intron (I-Zm.DnaK:1).

SEQ ID NO. 2 is the DNA sequence of the promoter (P-Zm.Cdc45-1:8).

SEQ ID NO. 3 is the DNA sequence of the leader sequence (L-Zm.Cdc45-1:1).

SEQ ID NO. 4 is a DNA sequence of EXP (EXP-Os.Cdc45-1:1:1) comprising a promoter (P-Os.Cdc45-1-1:1:1) operably linked to 5' of the leader sequence (L-Os.Cdc45-1-1:1:1).

SEQ ID NO. 5 is the DNA sequence of the promoter (P-Os.Cdc45-1-1:1:1).

SEQ ID NO. 6 is the DNA sequence of the leader sequence (L-Zm.Cdc45-1:1).

SEQ ID NO. 7 is the DNA sequence of EXP (EXP-at. Mei 1) consisting of a promoter and a leader sequence.

SEQ ID NO. 8 is the DNA sequence of the 3' UTR (T-at. Mei 1-1:2:1).

SEQ ID NO. 9 is the DNA sequence of EXP (EXP-At.Cdc45:1:1) comprising a promoter (P-At.Cdc45-1:1:1) operably linked to 5' of the leader sequence (L-At.Cdc45-1:1:1).

SEQ ID NO. 10 is the DNA sequence of the promoter (P-at. Cdc45-1:1:1:1).

SEQ ID NO. 11 is the DNA sequence of the leader sequence (L-At.Cdc45-1:1:1).

SEQ ID NO. 12 is the DNA sequence of the 3' UTR (T-At.Cdc45:1).

SEQ ID NO. 13 is the DNA sequence of EXP (EXP-at. Swi 1) consisting of a promoter and a leader sequence.

SEQ ID NO. 14 is the DNA sequence of EXP (EXP-at. Swi1a) consisting of a promoter and a leader sequence.

SEQ ID NO. 15 is the DNA sequence of the 3' UTR (T-At. Swi 1-1:2:1).

SEQ ID NO. 16 is a DNA sequence of EXP (EXP-At. Asy1:1:1) comprising a promoter (P-At. Asy1-1:1:1) operably linked to 5' of the leader sequence (L-At. Asy1-1:1:1).

SEQ ID NO. 17 is the DNA sequence of the promoter (P-At. Asy1-1:1:1).

SEQ ID NO. 18 is the DNA sequence of the leader sequence (L-At. Asy1-1:1:1).

SEQ ID NO. 19 is the DNA sequence of the 3' UTR (T-At. Asy1-1:1:1).

SEQ ID NO. 20 is a DNA sequence of EXP (EXP-Gm. Rsp-1:1) comprising a promoter (P-Gm. Rsp-1-1:1:1) operably linked to 5' of a leader sequence (L-Gm. Rsp-1-1:1:1).

SEQ ID NO. 21 is the DNA sequence of the promoter (P-Gm. Rsp-1-1:1:1).

SEQ ID NO. 22 is a DNA sequence of the leader sequence (L-Gm. Rsp-1-1:1:1).

SEQ ID NO. 23 is the DNA sequence of the 3' UTR (T-At.Cdc45:3).

SEQ ID NO. 24 is the DNA sequence of the 3' UTR (T-At.Cdc45:4).

SEQ ID NO. 25 is the DNA sequence of EXP (EXP-Gm. Rsp-1+Gm. Rsp-1+AtpE: 1) comprising a promoter (P-Gm. Rsp-1-1:1:1) operably linked to the 5 'of a leader sequence (L-Gm. Rsp-1-1:1:1) operably linked to the 5' of an intron (I-at. AtpE: 1).

SEQ ID NO. 26 is the DNA sequence of the intron (I-At. AtpE: 1).

SEQ ID NO. 27 is a DNA sequence of EXP (EXP-Zm.Cdc45-2+Zm.DnaK: 1: 2) comprising a promoter (P-Zm.Cdc45-2-1: 3) operably linked to 5 'of a leader sequence (L-Zm.Cdc45-2-1: 1) operably linked to 5' of an intron (I-Zm.DnaK: 1).

SEQ ID NO. 28 is the DNA sequence of the promoter (P-Zm.Cdc45-2-1:1:3).

SEQ ID NO. 29 is the DNA sequence of the leader sequence (L-Zm.Cdc45-2-1:1:1).

SEQ ID NO. 30 is the DNA sequence of EXP (EXP-Zm.Zm13:2) comprising a promoter (P-Zm.Zm13:2) operably linked to 5' of the leader sequence (L-Zm.Zm13:2).

SEQ ID NO. 31 is the DNA sequence of the promoter (P-Zm.Zm13:2).

SEQ ID NO. 32 is the DNA sequence of the leader sequence (L-Zm.Zm13:2).

SEQ ID NO. 33 is a DNA sequence of EXP (EXP-Zm.wall+Zm.DnaK: 1:5) comprising a promoter (P-Zm.wall-1:1:9) operably linked to 5 'of a leader sequence (P-Zm.wall-1:1:1) operably linked to 5' of an intron (I-Zm.DnaK: 1).

SEQ ID NO. 34 is the DNA sequence of the promoter (P-Zm.wall-1:1:9).

SEQ ID NO. 35 is the DNA sequence of the leader sequence (L-Zm.wall-1:1:1).

SEQ ID NO. 36 is the DNA sequence of EXP (EXP-Syn 1) consisting of a promoter and a leader sequence.

SEQ ID NO. 37 is the DNA sequence of EXP (EXP-Syn 1 a) consisting of a promoter and a leader sequence.

SEQ ID NO. 38 is the DNA sequence of the 3' UTR (T-At. Syn 1-1:2:1).

SEQ ID NO. 39 is the DNA sequence of the intron (I-Zm.DnaK: 1).

SEQ ID NO. 40 is the DNA sequence of EXP (EXP-At.Dmc1+Zm.DnaK: 1:1) comprising a promoter (P-At.Dmc1:1) operably linked to 5 'of a leader sequence (L-At.Dmc1-1:1:1) operably linked to 5' of an intron (I-Zm.DnaK: 1).

SEQ ID NO. 41 is the DNA sequence of the promoter (P-at. Dmc1:1).

SEQ ID NO. 42 is the DNA sequence of the leader sequence (L-at. Dmc1-1:1:1).

SEQ ID NO. 43 is the coding sequence of Cre-recombinase (Cre) with a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (Genbank accession number X04753).

SEQ ID NO. 44 is the DNA sequence of the Cre-recombinase site-specific recombination site (RS-P1. Lox1: 1).

SEQ ID NO. 45 is the DNA sequence of the Cre-recombinase site-specific recombination site (RS-P1. Lox. TA TA-R9-1:1:1).

SEQ ID NO. 46 is the DNA sequence of EXP (EXP-Os. Act1:1) consisting of the promoter, leader sequence and intron from the rice actin 1 gene.

SEQ ID NO. 47 is the coding sequence of plastid-targeted EPSPS (CP 4) which confers tolerance to the herbicide glyphosate.

SEQ ID NO. 48 shows the DNA sequence of the 3' UTR (T-AGRtu. Nos. 13).

SEQ ID NO. 49 is the DNA sequence of EXP (EXP-Os. Act1+CaMV.35S.2xA1-B3+Ta. Lhc B1:1) consisting of the enhanced promoter and leader sequence.

SEQ ID NO. 50 is a coding sequence for beta-Glucuronidase (GUS) having a processable intron derived from the potato light-induced tissue specific ST-LS1 gene (Genbank accession number X04753).

SEQ ID NO. 51 is the DNA sequence of the 3' UTR (T-St. Pis 4-1:4:1).

SEQ ID NO. 52 is the DNA sequence of EXP (EXP-Os. TubA-3:1) consisting of the promoter, leader sequence and intron from the rice tubulin gene.

SEQ ID NO. 53 is the DNA sequence of EXP (EXP-at. Act7:2) consisting of a promoter, a leader sequence and an intron derived from the Arabidopsis actin 7 gene.

SEQ ID NO. 54 is a plastid-targeted coding sequence of GOI-at. ShkG-CTP2+ec. AadA-SPC/STR 1:1, which confers resistance to the antibiotic spectinomycin.

SEQ ID NO. 55 is the DNA sequence of EXP (EXP-CaMV. 35S-enh: 1:2) consisting of the enhanced promoter and leader sequence.

SEQ ID NO. 56 is the DNA sequence of the promoter (P-Br. Snap 2-1:1:20).

SEQ ID NO. 57 is a DNA sequence encoding a chloroplast transit peptide (TS-Ps. RbcS-3C-1:3:1).

SEQ ID NO. 58 is a DN A coding sequence encoding the crtB gene (CR-PANag. CrtB. Nno-1:4:1).

SEQ ID NO. 59 is the DNA sequence of the 3' UTR (T-Br. Snap 2-1:3:6).

SEQ ID NO. 60 is the DNA sequence of EXP (EXP-vf. Usp88-enh: 1:1) consisting of an enhancer, a chimeric promoter (P-vf. Usp 88-chimera) and a leader sequence.

SEQ ID NO. 61 is the DNA sequence of the chimeric promoter P-vf. Usp88-chimera, consisting of the following elements: an enhancer derived from the vf. Usp88 promoter operably linked to 5' of the vf. Usp88 promoter.

SEQ ID NO. 62 is the DNA sequence of the leader sequence (L-vf. Usp-1:1:1).

SEQ ID NO. 63 is a DNA coding sequence encoding the splA gene (CR-AGRtu. SplA-C58:1:3).

SEQ ID NO. 64 is the DNA sequence of EXP (EXP-Gm. Nmh7:1) consisting of the following elements: a promoter (P-Gm.Nmh7-1:1:12) operably linked to 5' of the leader sequence (L-Gm.Nmh7:1).

SEQ ID NO. 65 is the DNA sequence of the promoter (P-Gm.Nmh7-1:1:12).

SEQ ID NO. 66 is the DNA sequence of the leader sequence (L-Gm.Nmh7:1).

SEQ ID NO. 67 is the DNA sequence of the 3' UTR (T-Gb.E6-3 b: 1:1).

Detailed Description

The present invention provides gene regulatory elements for driving the expression of site-specific recombinases in plants which will result in efficient automatic excision of marker gene cassettes. The invention also provides constructs and recombinant DNA molecules comprising the regulatory elements. The invention also provides methods of automatically excision of at least two transgene expression cassettes from the genome of a transgenic plant by using constructs comprising the transgene cassettes, wherein the gene regulatory elements described herein are operably linked to a site-specific recombinase gene.

The following definitions are provided for certain terms and phrases used herein. Unless otherwise defined in the present disclosure, terms and phrases used herein should be understood by those of ordinary skill and knowledge in the relevant art in light of their ordinary meaning.

Site-specific recombinase and excision of DNA segments

As used herein, a "site-specific recombinase" is an enzyme that binds to a particular DNA recognition sequence and catalyzes DNA cleavage, DNA strand exchange, and recombination of DNA between two site-specific recombinase site sequences. "site-specific recombination" or "site-specific recombinase system" or "site-specific recombinase technology" or "site-directed recombination" or "site-directed recombinase system" or "site-directed recombinase technology" describe various specialized recombination processes involving the exchange between defined DNA sites. As used herein, the term "flanking" refers to two or more sequences, such as site-specific recombination site sequences, located on either side of one or more specific loci/gene sites, genes, sequences, transgenes, or expression cassettes. Site-specific recombination site sequences can be cloned into a recombinant DNA construct relative to 5 'and 3' of the DNA segment comprising the expression cassette under which recombination will occur (i.e., flanking the segment of DNA). Depending on the initial arrangement of parent site-specific recombination sites, site-specific recombination has one of three possible outcomes: integration (insertion of foreign DNA segment), excision (removal of DNA segment) or inversion (rotation of DNA segment 180 degrees before re-ligating the two end segments). Integration results from recombination between sites on different DNA molecules (provided that at least one parent chromosome is circular) and occurs in a uniquely defined orientation.

For recombination sites located on the same DNA molecule or chromosome, the outcome may depend on their relative orientation. Although inversion of a DNA segment may result from exchange between inverted (head-to-head) sites, excision may result from recombination between sites in the head-to-tail direction (Nigel et al (2006) Mechanisms of Site-Specific recombination. Annu. Rev. Bioche m, 75:567-605). A number of Site-specific recombinases can be used to cleave DNA between two Site-specific recombinase recognition sites, such as Cre-recombinase recognizing Lox sites, flp-recombinase recognizing FR T sites (see, e.g., lyznik, L. Et al, (2000) Gene Transf er Mediated by Site-Specific Recombination Systems, plant Molecular Biology Manual N, 1-26), R-recombinase recognizing RS sites (see, e.g., machida, C. Et al, (2000) Use of the R-RS Site-Specific Recombination Sy stem in Plants, plant Molecular Biology Manual N2, 1-23), or Gin-recombinase recognizing GIX sites (see, e.g., maeser, S.et al, (1991) The Gin reco mbinase of phage Mu can catalyze Site-specific recombination in plant protoplasts, mol Genet, 230:170-176). Each of the above-described site-specific recombinase systems has been shown to function in plants. The Cre/Lox site-specific recombinase system is the most commonly relied upon marker excision system in plant biotechnology.

Site-specific recombinases can be used in plant biotechnology to remove marker gene expression cassettes as well as other expression cassettes and DNA segments from transgenic plants. Typically, plants are transformed with a recombinant DNA construct or vector comprising a plurality of expression cassettes. The expression cassettes can be used to express transgenes that provide beneficial properties to plants as well as transgenes that serve as markers to select transformed plant cells, such as antibiotic resistance genes, herbicide tolerance genes, or other transgenes useful in the selection process. The transgene cassette for the marker gene is flanked by a pair of site-specific recombinase recognition sites. Following transformation and selection, regenerated transformed plants are grown. Excision of the marker gene can then be removed by various hybridization strategies (by hybridization with a plant site-specific recombinase expression line or by automatic excision).

Hybridization using plant site-specific recombinase expression lines is generally performed as follows. Let R be ₀ The transformed plants were selfed. R is then selected for the presence of the recombinant DNA construct ₁ And (5) offspring plants. Then let the selected R ₁ Progeny plants are selfed and R is selected to be homozygous for the recombinant DNA construct insertion ₂ And (5) offspring plants. Then homozygously R ₂ The progeny plant is crossed to another line expressing the recombinase. As a result of this hybridization, the recombinase cleaves the marker gene cassette flanked by site-specific recombinase recognition sequences, producing F comprising the desired cassette but with the marker gene cassette excised from the genome ₁ And (5) offspring plants. Then let the obtained F ₁ Offspring were selfed and F was selected that lacks the recombinase but is homozygous for the now modified recombinant DNA construct insertion ₂ And (5) offspring plants.

Another strategy to remove marker gene expression cassettes is by means of automatic excision. Similar to the excision methods described above, the expressed recombinase is used to excision of the marker gene cassette, but rather than hybridizing the transformed plant to another line expressing the recombinase, the recombinase gene cassette is located in the same recombinant DNA construct and flanked by the site-specific recombinase site sequence and the marker gene cassette. The expression cassette that remains in the transgenic plant after automatic excision is expected to be present in the recombinant DNA construct outside the site-specific recombinase site sequence. After transformation and plant regeneration, R containing the recombinant DNA construct is generated ₀ And (5) a plant. Those R can then be made ₀ Plant selfing and the resulting R can be selected for the presence of altered recombinant DNA constructs in which the marker gene expression cassette and recombinase expression cassette have been excised ₁ And (5) offspring plants. An advantage of the automatic excision system is that one can eliminate the marker gene expression cassette by means of fewer generations than if a site-specific recombinase excision system were used in which hybridization to another line expressing the site-specific recombinase is required.

In automatic excision to produce label-free R ₁ One complicating factor in the development of progeny plants, automatic excision, is the finding of expression elements that provide for the expression of the site-specific recombinase at the correct time. One approach is to use expression elements that are active in the germ line or embryo phase of the plant, but not all germ line or embryo preferred expression elements will provide successful results for efficient automatic excision to occur. Some germline-preferred or embryo-preferred expression elements may have a leak in their expression, and some are unable to express the site-specific recombinase at sufficient levels to effectively excise the marker and/or recombinase gene. In addition, the germline or embryo expression elements may provide effective automatic excision only in specific crop species (such as corn, soybean, or cotton) but not in all three crops.

DNA molecules

As used herein, the term "DNA" or "DNA molecule" refers to a double-stranded DNA molecule of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide bases or a DNA molecule. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule read from the 5 '(upstream) end to the 3' (downstream) end.

As used herein, a "recombinant DNA molecule" or "recombinant DNA construct" is a DNA molecule or construct, respectively, that comprises a combination of DNA sequences that do not naturally occur together without human intervention. For example, a recombinant DNA molecule may comprise at least two DNA sequences that are heterologous to each other: DNA sequences derived from DNA sequences present in nature, synthetic DNA sequences, and/or DNA sequences that have been incorporated into the genomic DNA of a host cell by genetic transformation, genome editing, or site-specific integration.

In this application, reference to an "isolated DNA molecule" or equivalent term or phrase is intended to mean that the DNA molecule is one that exists alone or in combination with other components but is not present in its natural environment. For example, a nucleic acid element that naturally occurs in the genome of an organism, such as a coding sequence, an intron sequence, an untranslated sequence, a leader sequence, a promoter sequence, a transcription termination sequence, etc., is not considered "isolated" as long as the element is native to the genome of the organism and in a location within the genome where it naturally occurs. However, each of these elements, as well as sub-portions of these elements, will be "isolated" within the scope of this disclosure as long as the elements are not within their native genome and/or are in their naturally occurring locations within the genome. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether it is present within a plasmid or similar vector used to transform the cell within the genome of the plant or bacterium, or is present in a detectable amount in a tissue, progeny, biological sample, or commodity product derived from the plant or bacterium.

As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. By aligning two sequences, e.g., a reference sequence and another sequence, to maximize the number of nucleotide matches in a sequence alignment with the appropriate internal nucleotide insertions, deletions, or gaps, an optimal sequence alignment of the two sequences is produced. As used herein, the term "reference sequence" may refer to a DNA sequence comprising one or more of SEQ ID NOS 1-26, 59-62 and 64-66.

As used herein, the term "percent sequence identity" or "percent identity" is the identity score of two optimally aligned sequences multiplied by 100. The "identity score" of a sequence optimally aligned to a reference sequence is the number of nucleotide matches in the optimal alignment divided by the total number of nucleotides in the reference sequence (i.e., the total number of nucleotides in the entire reference sequence over the entire length). Thus, some embodiments of the present disclosure provide a DNA molecule comprising a regulatory sequence having at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to a reference sequence, such as one of SEQ ID NOs 1-26, 59-62, and 64-66, when optimally aligned with said reference sequence. According to embodiments of the invention, the regulatory sequences may be operably linked to a transcribable DNA sequence that may encode a site-specific recombinase.

Regulatory element

Regulatory elements such as promoters, leader sequences (also known as 5 'UTRs), enhancers, introns, and transcription termination regions (or 3' UTRs) play an indispensable role in the overall expression of genes in living cells. As used herein, the term "regulatory element" refers to a DNA molecule or sequence or DNA segment having gene regulatory activity. As used herein, the term "gene regulatory activity" refers to the ability to affect expression of an operably linked transcribable DNA molecule, for example by affecting transcription and/or translation of the operably linked transcribable DNA molecule. Regulatory elements that function in plants, such as promoters, leader sequences, enhancers, introns and 3' utrs, may be used to modify plant phenotypes by genetic engineering. According to an embodiment of the disclosure, the regulatory element is a promoter having a sequence comprising SEQ ID No. 2, 5, 10, 17, 21, 61 or 65, or a sequence having at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity to SEQ ID No. 2, 5, 10, 17, 21, 61 or 65, or a functional fragment or portion of any of the foregoing sequences. According to an embodiment of the present disclosure, the regulatory element is a leader sequence having a sequence comprising SEQ ID NO 3, 6, 11, 18, 22, 61 or 66, or a sequence having at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity to SEQ ID NO 3, 6, 11, 18, 22, 61 or 66, or a functional fragment or portion of any of the foregoing sequences that can affect expression of an operably linked transcribable DNA sequence.

As used herein, a "fragment" of a promoter (or promoter sequence) or regulatory element comprises a fragment or portion of a promoter (or promoter sequence) or regulatory element, respectively, and a "functional fragment" of a promoter (or promoter sequence) or regulatory element comprises a fragment or portion of a promoter (or promoter sequence) or regulatory element, respectively, that affects, regulates or drives expression of an operably linked transcribable DNA sequence. According to some embodiments, a "functional fragment" of a promoter (or promoter sequence) or regulatory element affects, modulates or drives expression of an operably linked transcribable DNA sequence in a similar manner to a promoter (or promoter sequence) or regulatory element.

As used herein, a "set of regulatory expression elements" or "EXP" sequence refers to a set of two or more operably linked regulatory elements, such as enhancers, promoters, leader sequences, and introns. Such two or more operably linked regulatory elements may typically be present together in the same construct and each operably linked to a transcribable DNA sequence. For example, a set of regulatory expression elements may consist of, for example, a promoter operably linked to the 5' of the leader sequence. EXP used to practice embodiments of the present invention can comprise SEQ ID NO:1, 4, 7, 9, 13, 14, 16, 20, 25, 60, or 64, and sequences having at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO:1, 4, 7, 9, 13, 14, 16, 20, 25, 60, or 64.

Regulatory elements may be characterized by their associated gene expression patterns in plants, plant tissues and plant cells, for example, their positive and/or negative effects on expression, such as constitutive expression or specific expression patterns, such as temporal, spatial, developmental, tissue, environmental, physiological, pathological or cell cycle expression, and/or chemical reaction or inducible expression, and any combination thereof, as well as quantitative or qualitative indications or patterns of expression. As used herein, a "gene expression pattern" is any pattern of transcription of an operably linked DNA molecule into a transcribed RNA molecule that results in the relative levels and abundance of the transcribed RNA molecule in various plant tissues and cells during development. Regulatory elements may include enhancers, promoters, leader sequences, 5 'UTRs, introns and/or 3' UTRs. Regulatory elements of the present disclosure may include regulatory elements or promoters of germline preference or embryo preference.

As used herein, the term "promoter" generally refers to a DNA molecule, segment, or sequence that is involved in the recognition and binding of RNA polymerase II and other proteins (such as trans-acting transcription factors) to initiate or regulate transcription. The promoter may be initially isolated upstream or 5 'untranslated region (5' UTR) of the genomic copy of the gene. Alternatively, the promoter may be a synthetically produced or engineered DNA molecule. Promoters may also be chimeric. Chimeric promoters are produced by fusion of two or more heterologous DNA molecules. Promoters useful in practicing embodiments of the present invention may include promoter elements comprising SEQ ID NOs 2, 5, 10, 17, 21, 61 or 65; or within any one of SEQ ID NOs 1, 4, 7, 9, 13, 14, 16, 20, 25, 60 and 64; or a sequence having at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID No. 2, 5, 10, 17, 21, 61, or 65; or a sequence having at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to a sequence within any one of SEQ ID nos. 1, 4, 7, 9, 13, 14, 16, 20, 25, 60, and 64; or a functional fragment or portion of any of the foregoing sequences. In particular embodiments, a DNA molecule as described herein, and any variant, fragment, portion or derivative thereof, is further defined as comprising promoter activity, i.e., being capable of acting as a promoter in a host cell, such as in a transgenic plant. In still further embodiments, a fragment of a promoter sequence may be defined as exhibiting promoter activity possessed by the starting promoter molecule from which it is derived, or the fragment may comprise a "minimal promoter" that provides basal levels of transcription and is composed of a TATA box or equivalent DNA sequence for identifying and binding to RNA polymerase II complexes for initiating transcription.

In one embodiment, fragments of the promoter sequences disclosed herein are provided. Promoter fragments may comprise promoter activity as described above and may be used alone or in combination with other promoters and/or promoter fragments, for example, for the construction of chimeric promoters, or in combination with other expression or regulatory elements and expression or regulatory element fragments. In particular embodiments, there is provided a promoter fragment comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 or more contiguous nucleotides of a promoter, promoter sequence, or DNA molecule having promoter activity as disclosed herein.

For example, modifying or altering expression using methods known in the art, such as by removing elements or element portions or nonfunctional spacer sequences that may have a positive or negative effect on expression; replicating elements that have a positive or negative effect on expression; inserting elements that have a positive or negative effect on expression; and/or replication or removal of elements having tissue-specific, developmental or cell-specific effects on expression, recombinant DNA molecules or constructs comprising promoter or regulatory elements derived from any of the promoter elements provided as SEQ ID NOs 2, 5, 10, 17, 21, 61 and 65, or from any sequence within any of SEQ ID NOs 1, 4, 7, 9, 13, 14, 16, 20, 25, 60 and 64, such as internal or truncated sequences or sequences with 5' deletions, may be produced. The enhancer element can be made, for example, using any recombinant DNA construct or molecule comprising a promoter or regulatory element derived from any promoter element provided as SEQ ID No. 2, 5, 10, 17, 21, 61 or 65, or from any sequence within SEQ ID No. 1, 4, 7, 9, 13, 14, 16, 20, 25, 60 or 64, including 3' deletions, wherein the TATA box element or equivalent sequence thereof and downstream sequences are removed. Further deletions may be made to remove any positive or negative effects on expression; tissue specificity; cell specificity; or time-specific (such as, but not limited to, circadian rhythm or developmental time) acting elements. Any promoter element provided as or contained within any one of SEQ ID NOs.2, 5, 10, 17, 21, 61 and 65, as well as fragments or enhancers derived therefrom, may be used to prepare chimeric transcription regulatory element compositions.

According to the invention, promoters or promoter fragments may be analyzed for the presence of known promoter elements (i.e., DNA sequence features such as TATA boxes and other known transcription factor binding site motifs). The skilled artisan can use the identification of such known promoter elements to design promoter variants having similar expression patterns to the original promoter.

As used herein, a "germline preferred promoter" is defined as a promoter that drives expression of an operably linked gene (or transgene) primarily in one or more germline cells of a plant but may also drive expression of an operably linked gene (or transgene) in other cells or tissues of a plant. "seed line" is used as a collective term for these cells, which are gamete cells or gamete cell progenitors, differentiate into gamete cells or gamete cell progenitors, or have at least one progeny that is gamete cells or gamete cell progenitors. Genetic modifications in germ line cells can be transferred to progeny plants by gametes derived or inherited from such germ line cells. The use of a germline preferred promoter to drive expression of a site-specific recombinase allows removal of markers and/or recombinase genes flanking the site-specific recombinase recognition sequence in the germline cell in which the site-specific recombinase is expressed. The resulting gametes will have an altered transgene in which the marker gene expression cassette and/or recombinase expression cassette are no longer present, and such altered transgene can then be transferred to the progeny plant. By using Germ line preferred promoters to drive expression of site-specific recombinases for automatic excision can be found in the gene from R ₀ R of parent plant for selfing or outcrossing ₁ Removal of the marker and/or recombinase gene is accomplished in the generation, as opposed to the next generation in the case where the site-specific recombinase may be introduced by hybridization with a different system having a recombinase expression construct.

The germ line preferred promoters that effectively drive automatic excision in plants are not identical, depending on the type of crop in which the excision is expected. For example, while the Arabidopsis DCM1 promoter has been shown to drive excision of the GUS marker gene in Arabidopsis germline cells (see, e.g., van Ex et al (2009) "Evaluation of seven promoters to achieve germline directed Cre-lox recombination in Arabidopsis thaliana" Plant Cell Rep.28:1509-1520), such a promoter does not drive automatic excision in stably transformed soybean plants (see, e.g., example 4 below). Other Arabidopsis derived germline preferred promoters may drive automatic excision in transgenic soybean plants, such as those comprising the promoter sequence of SEQ ID NO. 10 or SEQ ID NO. 17, as well as those within SEQ ID NO. 7, SEQ ID NO. 13 or SEQ ID NO. 14 (see, e.g., example 3). Promoters derived from the plant-derived germ-line-preferred CDC45 gene have been shown to be capable of driving automatic excision in crop plants. The Arabidopsis CDC45 promoter SEQ ID NO 10 was shown to drive efficient automatic excision in transgenic soybean (example 3) and cotton plants (example 5). The CDC45-1 promoter of maize (SEQ ID NO: 2) and rice (SEQ ID NO: 5) was shown to drive efficient automatic excision in transgenic maize plants (example 2). Although the soybean Rsp1 promoter (SEQ ID NO: 21) is shown to drive automatic excision in soybean plants, the Rsp1 promoter requires an intron (I-at. AtpE:1, SEQ ID NO: 26) operably linked to the Rsp1 promoter and leader sequence to drive efficient automatic excision in transgenic cotton plants. However, operably linking the I-at.atpe intron to the Rsp1 promoter and leader sequence failed to drive efficient automatic excision in transgenic soybean. It was shown that the soybean Rsp1 promoter and P-Gm.Nmh7-1:1:12 (SEQ ID NO: 65) driven automatic excision of the four transgene cassettes: cre-recombinase; marker gene, cpf1 and guide RNA expression cassette.

As used herein, an "embryo-preferred promoter" is defined as a promoter that drives expression of an operably linked gene (or transgene) primarily in one or more cells of a embryo but may also drive expression of an operably linked gene (or transgene) in other cells or tissues of a seed or plant. A chimeric promoter P-vf. Usp 88-chimera (SEQ ID NO: 61) showing embryo preference driven automatic excision in transgenic canola plants.

As used herein, the term "leader sequence" refers to a DNA molecule isolated from the untranslated 5 'region (5' utr) of a gene, and is generally defined as a segment of nucleotides between a Transcription Start Site (TSS) and a protein coding sequence start site. Alternatively, the leader sequence may be a synthetically produced or engineered DNA element. The leader sequence may be used as a 5' regulatory element for regulating expression of the operably linked transcribable DNA sequence. The leader sequence may be used with a heterologous promoter or its native promoter. Leader sequences for practicing embodiments of the invention may include SEQ ID NOs 3, 6, 11, 18, 22, 62 or 66; or a sequence having at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID No. 3, 6, 11, 18, 22, 62, or 66; or a functional fragment or portion of any of the foregoing sequences; or any leader element contained within any of SEQ ID NOs 1, 4, 7, 9, 13, 14, 16, 20, 25, 60 and 64 or within any sequence having at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity to SEQ ID NOs 1, 4, 7, 9, 13, 14, 16, 20, 25, 60 and 64, or a functional fragment or portion thereof. In particular embodiments, such DNA sequences may be defined as being capable of acting as leader sequences in host cells, including, for example, transgenic plant cells. In one embodiment, such sequences are defined as comprising leader sequence activity.

Leader sequences (also referred to as 5' UTRs) presented as SEQ ID NOs 3, 6, 11, 18, 22, 62 and 66; or any leader element contained within any of SEQ ID NOs 1, 4, 7, 9, 13, 14, 16, 20, 25, 60 and 64 may be comprised of regulatory elements and/or may employ secondary structures that regulate or affect transcription or translation of operably linked transcribable DNA sequences. Leader sequences presented as SEQ ID NOs 3, 6, 11, 18, 22, 62 and 66 or any fragment thereof may be made according to the present disclosure; or any leader sequence element contained within any one of SEQ ID NOs 1, 4, 7, 9, 13, 14, 16, 20, 25, 60 and 64 or any fragment thereof, to produce chimeric regulatory elements that affect transcription or translation of an operably linked transcribable DNA sequence.

As used herein, the term "intron" refers to a DNA molecule or sequence that can be isolated or identified from a gene and can be generally defined as a region that is spliced out during the processing of pre-translational messenger RNA (mRNA). Alternatively, the introns may be synthetically produced or engineered DNA elements. Introns may contain enhancer elements that affect transcription of an operably linked gene or a transcribable DNA sequence. Introns may be used as regulatory elements for regulating expression of operably linked transcribable DNA sequences. A construct may comprise introns, and the introns may or may not be heterologous with respect to the transcribable DNA sequence. Examples of introns in the art include the rice actin intron and the maize HSP70 intron.

In plants, the inclusion of some introns in the genetic construct results in increased mRNA and protein accumulation relative to constructs lacking introns. This effect is known as "intron-mediated enhancement" (IME) of gene expression. Introns known to stimulate expression in plants have been identified in maize genes (e.g., tubA1, adh1, sh1, and Ubi 1), rice genes (e.g., tpi), and dicotyledonous plant genes such as those from petunia (e.g., rbcS), potato (e.g., st-ls 1), and from arabidopsis thaliana (e.g., ubq3 and pat 1). Deletions or mutations within the splice sites of introns have been shown to reduce gene expression, suggesting that splicing may be required for IME. However, IME in dicots has been shown by point mutations within the splice site of the pat1 gene from Arabidopsis thaliana. The same intron has been shown to exhibit disadvantages for multiple uses in one plant. In those cases, it is necessary to collect the basic control elements to construct the appropriate recombinant DNA elements. Exemplary introns for use in practicing the invention are presented as SEQ ID NOs 26 and 39.

As used herein, the term "3 'transcription termination sequence", "3' untranslated region" or "3'utr" refers to a DNA sequence transcribed into an untranslated region within the 3' portion of an mRNA molecule, as is commonly understood in the art. The 3 'untranslated region of an mRNA molecule can be generated by specific cleavage and 3' polyadenylation (also known as polyA tail formation). The 3' utr may be operably linked to and downstream of the RNA or protein encoding portion of the transcribable DNA sequence and may include polyadenylation signals and other regulatory elements or signals capable of affecting transcription, mRNA processing, and/or gene expression. PolyA tails are thought to play a role in mRNA stability and translation initiation. Examples of 3 'transcription termination molecules in the art are the nopaline synthase 3' region, the wheat hsp17 'region, the pea rubisco small subunit 3' region, the cotton E6 3 'region and the coixoxin 3' UTR.

The 3' UTR generally finds advantageous use in the recombinant expression of specific DNA molecules. A weak 3' utr may create readthrough, which may affect the expression of DNA molecules located in adjacent expression cassettes. Proper control of transcription termination may prevent read-through into downstream located DNA sequences (e.g., other expression cassettes), and may further allow efficient recycling of RNA polymerase to increase gene expression. Efficient termination of transcription (release of RNA polymerase II from DNA) is a prerequisite for re-initiation of transcription, thus directly affecting overall transcription levels. Upon termination of transcription, mature mRNA is released from the synthesis site and the template is transported into the cytoplasm. Eukaryotic mRNA accumulates in vivo in the form of poly (A), making it difficult to detect transcription termination sites by conventional methods. However, it is difficult to predict functional and effective 3'UTRs by bioinformatics methods, as there is no conserved DNA sequence that allows for easy prediction of effective 3' UTRs.

Modulation of gene function by 3'utrs is a relatively new area, since only recent sequencing technologies provide us with a panoramic view of 3' utrs across species and cell types. Detailed functional and mechanistic studies were performed on only a few 3' utr models before sequencing technology was available. While these model 3'utrs make a significant contribution to our understanding of 3' utr biology, conclusions regarding their regulatory function are limited and more focused on mRNA stability. Computer analysis of the genome-wide region (Mayr, christine (2017) Regulation by 3'-Untranslated regions. Annual Review of Genetics, 51:171-194) revealed that motifs in the 3' UTR were predominantly conserved on one strand, consistent with the role of 3'UTR in regulating gene expression at the posttranscriptional level (Xie, X. Et al, (2005) Systematic discovery of regulatory motifs in human promoters and 3'UTRs by comparison of several mammals,Nature 434:338-345). The 3' utr determines protein levels by regulating mRNA stability and translation mediated primarily by AU-rich elements and mirnas. The 3' UTR also achieves local translation by modulating mRNA localization. The length of the 3' UTR can be regulated by selective cleavage and polyadenylation. The 3' utr mediates protein-protein interactions (PPI), with a broad impact on protein complex formation, protein localization and protein function. The 3' UTR regulates gene expression by binding to RNA Binding Proteins (RBPs). RBP binds to the 3'utr cis element and mediates 3' utr function by recruiting effector proteins. The RBP cooperates with other bound RBPs to achieve in vivo functional specificity. The composition of RBPs that bind to the 3' utr at a given moment is dynamic and can vary depending on the local environment, for example, by adding post-translational modifications, local expression of other RBPs, and interactions with membrane and cytoskeletal wires. RBP binding is also affected by the formation of secondary and tertiary RNA structures that regulate accessibility of the 3'UTR (Mayr, christine (2017) Regulation by 3' -Untranslipated regions. Annual Review of Genetics, 51:171-194).

A series of adenosine bases are added to the 3' end of the RNA molecule to produce a poly (A) tail. This provides the mR NA with binding sites for a class of regulatory factors known as poly (a) binding proteins (PABPs), which play a role in the regulation of gene expression, including mRNA export, stability and decay, and translation. The 5' cap structure of the mRNA and the poly-A tail act synergistically to control mRNA translation. Binding of PABP to the poly (a) tail promotes interaction with eIF4F bound to the 5' cap structure, resulting in mRNA cyclization that promotes translation initiation and ensures ribosome recycling and efficient translation. This interaction also allows inhibition of translation by inhibitor proteins that bind to the 3' UTR (Barret, L et al (2012) Regulation of eukaryotic gene ex pression by the untranslated regions and other non-coding elements.cell.mol.Life Sci.69:3613-3634).

From a practical point of view, it is often beneficial that the 3' UTR used in the expression cassette has the following characteristics. First, the 3' UTR should be able to terminate transcription of the transgene efficiently and effectively and prevent the transcription from being read through into any adjacent DNA sequence that may consist of one expression cassette if the cassettes are located in another, or into adjacent chromosomal DNA in which the constructs are inserted. Second, the 3' UTR should not cause a decrease in transcriptional activity conferred by promoters, leader sequences, enhancers and introns used to drive expression of the DNA sequence. Finally, in plant biotechnology, the 3' utr is commonly used to initiate an amplification reaction of reverse transcribed RNA extracted from transformed plants, and to: (1) Once the expression cassette is integrated into the plant chromosome, its transcriptional activity or expression is assessed; (2) assessing the number of copies inserted in the plant DNA; and (3) evaluating zygosity of the resulting seeds after breeding. The 3' UTR is also used in amplification reactions of DNA extracted from transformed plants to characterize the integrity of the insert cassette. The 3' UTRs useful in the practice of the present invention are presented as SEQ ID NOs 12, 15, 19, 23, 24 and 59.

As used herein, the term "chimeric" refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, wherein neither the first nor the second DNA molecule is typically found in this configuration (i.e., fused to another). Thus, chimeric DNA molecules are novel DNA molecules that are not normally found in nature. As used herein, the term "chimeric promoter" refers to a promoter produced by such manipulation of a DNA molecule. Chimeric promoters may combine two or more DNA fragments, e.g., a promoter fused to an enhancer element. Thus, the invention includes the design, construction and use of chimeric promoters for regulating expression of operably linked transcribable DNA molecules according to the methods disclosed herein. The chimeric promoter used in the practice of the present invention is presented as SEQ ID NO. 61.

Chimeric regulatory elements can be designed to comprise various constituent elements that can be operably linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation-independent cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of regulatory elements, among other methods known in the art. The resulting various chimeric regulatory elements may consist of the same constituent elements or variants of the same constituent elements, but differ in terms of DNA sequence or DNA sequences comprising linked DNA sequences or sequences allowing the operative linkage of the constituent parts. In the present invention, the DNA sequences as provided in SEQ ID NOS.1-26, 59-62 and 64-66 may provide regulatory element reference sequences, wherein the constituent elements comprising said reference sequences may be linked by methods known in the art and may comprise substitutions, deletions and/or insertions or mutations of one or more nucleotides naturally occurring in bacterial and plant cell transformation.

As used herein, the term "variant" refers to a second DNA molecule, such as a regulatory element, which is similar but not identical in composition to the first DNA molecule, and wherein the second DNA molecule still retains the general function of the first DNA molecule, i.e. the same or similar expression pattern, e.g. by more or less equivalent transcriptional activity. The variant may be a shorter or truncated form of the first DNA molecule, or a variant of the sequence of the first DNA molecule, such as a variant having different restriction enzyme sites and/or internal deletions, substitutions or insertions. A "variant" may also encompass regulatory elements having a nucleotide sequence comprising a substitution, deletion or insertion of one or more nucleotides of a reference sequence, wherein the derived regulatory element has more or less or equivalent transcriptional or translational activity than the corresponding parent regulatory molecule. Regulatory element "variants" will also include variants resulting from mutations that occur naturally in bacterial and plant cell transformation. In the present invention, the polynucleotide sequences provided as SEQ ID NOS.1-26, 59-62 and 64-66 can be used to produce variants that are similar in composition but not identical to the DNA sequence of the original regulatory element, but which still retain the general function (i.e., the same or similar expression pattern) of the original regulatory element. The creation of such variants of the invention is well within the ordinary skill in the art in light of this disclosure and is included within the scope of the invention.

As used herein, a "transcribable DNA sequence" is any DNA sequence that, when operably linked to a promoter, can be transcribed into RNA. A transcribed RNA molecule encoded by a transcribable DNA sequence operably linked to regulatory elements provided herein may be translated to produce a protein molecule or may provide an antisense or other functional or regulatory RNA molecule, such as a double-stranded hairpin RNA (dsRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA), a microrna (miRNA), a small interfering RNA (siRNA), etc.

As used herein, the term "protein expression" is any pattern of translation of a transcribed RNA molecule into a protein molecule. Protein expression is characterized by its temporal, spatial, developmental or morphological properties, as well as its quantitative or qualitative indications or expression patterns.

The efficacy of modifications, duplications or deletions described herein in terms of the desired expression of a particular transgene can be empirically tested in stable and transient plant assays (such as those described in the working examples herein), thereby validating the results, which can vary depending on the changes made in the starting DNA molecule and the purpose of the changes.

Constructs

As used herein, the term "construct" refers to any DNA molecule or vector, or segment or portion of a DNA molecule, vector or chromosome, that is derived from any one or more sources and that is capable of transfection or genomic integration, comprising at least two DNA sequences that are functionally operably linked to each other. For example, a construct may comprise two operably linked sequences, such as a regulatory element or promoter operably linked to a coding sequence or a transcribable DNA sequence. The construct may be a recombinant DNA construct. An example of a construct that is a linear recombinant DNA segment is T-DNA. As used herein, "vector" refers to a DNA molecule, such as a plasmid, cosmid, virus, phage, or other linear or circular DNA molecule, that may contain or comprise a construct of the present disclosure, and "DNA transformation vector" refers to any DNA molecule or vector that comprises a recombinant DNA construct that can be used for transformation purposes-i.e., for introducing the recombinant DNA molecule or construct into a host cell, such as a plant cell. According to some embodiments, the DNA transformation vector may comprise T-DNA fragments bounded by left and/or right border sequences, which may be used for bacterial-mediated transformation, such as rhizobia-mediated or agrobacterium-mediated transformation. Constructs typically include one or more expression cassettes, a gene coding sequence operably linked to one or more regulatory sequences such as a promoter, or a transcribable DNA sequence. As used herein, an "expression cassette" refers to a DNA sequence comprising at least one transcribable DNA sequence operably linked to one or more regulatory elements (typically at least one promoter and one 3' utr).

As used herein, the term "operably linked" refers to a functional relationship between two or more physically linked DNA sequences comprising a first and a second DNA sequence arranged such that the first DNA sequence affects the function or expression of the second DNA sequence. The two DNA sequences may or may not be part of a single contiguous DNA molecule, and may or may not be contiguous. For example, a promoter is operably linked to a transcribable DNA sequence if the promoter regulates the transcription of the transcribable DNA sequence of interest in a cell. For example, a leader sequence is operably linked to a transcribable DNA sequence when it is capable of affecting the transcription or translation of the DNA sequence.

In one embodiment, the constructs of the invention may be provided as dual tumor-inducing (Ti) plasmid border constructs having right (RB or agrtu. RB) and left border (LB or agrtu. LB) regions of Ti or Ri plasmids isolated from agrobacterium species (e.g., agrobacterium tumefaciens or agrobacterium rhizogenes) comprising T-DNA that allows integration of the T-DNA into the genome of a plant cell along with the transfer molecules provided by the agrobacterium cells (see, e.g., U.S. Pat. No. 6,603,061). Constructs may also contain a plasmid backbone DNA segment that provides replication functions and antibiotic selection in bacterial cells, e.g., an escherichia coli origin of replication, such as ori322; a broad host range origin of replication, such as oriV or oriRi (see, e.g., ye et al, transgenic Research (4): 773-86, 2011); and coding regions for selectable markers, such as Spec/Strp encoding Tn7 aminoglycoside adenylate transferase (aadA) which confers resistance to spectinomycin or streptomycin; or gentamicin (Gm, gent) selectable marker gene. For plant transformation, the host bacterial strain is typically Agrobacterium tumefaciens ABI, C58 or LBA4404, however, other strains known to those skilled in the art of plant transformation may also function in the present invention.

Methods for assembling constructs and introducing them into cells in such a way that a transcribable DNA molecule is transcribed into a functional mRNA molecule, which is translated and expressed as a protein, are known in the art. Conventional compositions and methods for making and using constructs and host cells are well known to those of skill in the art for practicing the present invention. Typical vectors for expressing nucleic acids in higher plants are well known in the art and include vectors derived from the Ti plasmid of Agrobacterium tumefaciens and pCaMVCN transfer control vectors.

Various regulatory elements may be included in the construct, including any of those provided herein. Any such regulatory element may be provided in combination with other regulatory elements. Such combinations may be designed or modified to produce desired regulatory features. In one embodiment, the construct of the invention comprises at least one regulatory element operably linked to a transcribable DNA molecule operably linked to a 3' utr.

Constructs of the invention may include any promoter or leader sequence provided herein or known in the art. For example, the promoters of the invention may be operably linked to a heterologous, non-translated 5' leader sequence, such as a leader sequence derived from a heat shock protein gene. Alternatively, the leader sequence of the present invention may be operably linked to a heterologous promoter, such as the cauliflower mosaic virus 35S transcript promoter.

The expression cassette may also include a transit peptide coding sequence encoding a peptide useful for subcellular targeting of an operably linked protein, particularly to a chloroplast, leucoplast, or other plastid organelle; mitochondria; a peroxisome; cavitation; or an extracellular location. Many chloroplast-localized proteins are expressed from nuclear genes in a precursor fashion and are targeted to the chloroplast by a Chloroplast Transit Peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, those proteins associated with the small subunit of ribulose-1, 5-bisphosphate carboxylase (SSU), ferredoxin oxidoreductase, light harvesting complex protein I and protein II, thioredoxin F, enolpyruvylshikimate phosphate synthase (EPSPS). Chloroplast transit peptides are described, for example, in U.S. patent No. 7,193,133. It has been demonstrated that non-chloroplast proteins can be targeted to chloroplasts by expression of heterologous CTPs operably linked to transgenes encoding the non-chloroplast proteins.

Transcribable DNA sequences

As used herein, the term "transcribable DNA sequence" refers to any DNA sequence capable of being transcribed into an RNA molecule, including, but not limited to, those having protein coding sequences and those that produce an RNA molecule having sequences for gene suppression. The type of DNA sequence may include, but is not limited to, a DNA sequence from the same plant, a DNA sequence from another plant, a DNA sequence from a different organism, or a synthetic DNA sequence, such as a DNA sequence containing antisense information to a gene, or a DNA sequence encoding a transgene in an artificial, synthetic, or other modified form. Exemplary transcribable DNA sequences for incorporation into constructs of the invention include, for example, DNA sequences or genes from a different species than the species into which the DNA sequences are incorporated, or genes derived from or present in the same species but integrated into recipient cells by genetic engineering methods other than classical breeding techniques.

By "transgene" is meant a transcribable DNA sequence that is heterologous to the host cell, at least in terms of its location in the host cell genome, and/or a transcribable DNA sequence that is artificially incorporated into the cell genome of the current or any previous generation host cell.

Regulatory elements, such as promoters of the invention, may be operably linked to a transcribable DNA sequence that is heterologous with respect to the regulatory element. As used herein, the term "heterologous" refers to a combination of two or more DNA molecules, where such a combination is not typically found in nature. For example, the two DNA molecules may be from different species and/or the two DNA molecules may be from different genes, e.g., different genes from the same species or the same gene from different species. Thus, if such a combination is not normally found in nature, the regulatory element is heterologous with respect to the operably linked transcribable DNA molecule, i.e., the sequence of the transcribable DNA molecule is not naturally operably linked to the regulatory element. By "heterologous transcribable DNA sequence" is meant that the transcribable DNA sequence is heterologous with respect to the polynucleotide sequence to which it is operably linked.

The transcribable DNA sequence may generally be any DNA sequence that requires expression of the transcript. Such expression of transcripts can lead to translation of the resulting mRNA molecules, resulting in protein expression. Alternatively, for example, a transcribable DNA sequence may be designed to ultimately result in reduced expression of a particular gene or protein. In one embodiment, this may be accomplished by using a transcribable DNA sequence oriented in the antisense orientation. The use of such antisense techniques is familiar to those of ordinary skill in the art. Any gene can be negatively regulated in this manner, and in one embodiment, the transcribable DNA sequence can be designed to inhibit a specific gene by expression of dsRNA, siRNA or miRNA molecules.

Thus, one embodiment of the invention is a recombinant DNA molecule comprising a regulatory element of the invention, such as those provided as SEQ ID NOS 1-26, 59-62 and 64-66, or fragments thereof, or a sequence or fragment thereof having at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity to any one of SEQ ID NOS 1-26, 59-62 and 64-66, so as to modulate transcription of said transcribable DNA sequence at a desired level or in a desired pattern upon integration of said construct in the genome of a transgenic plant cell. In one embodiment, the transcribable DNA sequence comprises a protein coding region of a gene, while in another embodiment, the transcribable DNA sequence comprises an antisense region of a gene or any other transcribable DNA sequence that causes inhibition of a specific target gene.

Genes of agronomic interest

The transcribable DNA sequence may be an agronomically advantageous gene. As used herein, the term "agronomically beneficial gene" or "agronomically beneficial transgene" refers to a transcribable DNA sequence that confers a desired characteristic or trait when expressed in a particular plant tissue, cell or cell type. The agronomically beneficial gene or transgene product may act in plants to affect plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease or pest resistance and/or environmental or chemical tolerance, or may act as a pesticide in the diet of the plant-feeding pests. In one embodiment of the invention, the regulatory element of the invention is incorporated into a construct such that the regulatory element is operably linked to a transcribable DNA sequence, which is an agronomically beneficial gene or transgene. In transgenic plants containing such constructs, the expression of genes of agronomic interest may confer beneficial agronomic traits. Beneficial agronomic traits may include, for example, but are not limited to, herbicide tolerance, insect control, improved or increased yield, disease resistance, pathogen resistance, improved plant growth and development, improved starch content, improved oil content, improved fatty acid content, improved protein content, improved fruit maturity, enhanced animal and human nutrition, biopolymer production, environmental stress tolerance or resistance, pharmaceutical peptides, improved processing quality, improved flavor, hybrid seed production utility, improved fiber production, and ideal biofuel production.

Non-limiting examples of agronomically beneficial genes (or transgenes) known in the art include those directed against: herbicide resistance (U.S. patent No.; a first number; 6,248,876; increased yield (U.S. Pat. No. 5,633,435, and 5,463,175), increased yield (U.S. Pat. No. USRE; first; second; first; second; and first), insect control (U.S. Pat. No. 5; first; 6,620,988; first; second; third; second; first; second), second; third; second; fourth; fifth, fourth; fifth, fifth; fifth, fourth; fifth, first, second, fifth, fifth, improved fatty acid content (U.S. patent no; a first number; a first number; number, and number), high protein yield (U.S. patent number), fruit maturity (U.S. patent number), enhanced animal and human nutrition (U.S. patent number, and 6,171,640), biopolymers (U.S. patent USRE number, and number), environmental stress resistance (U.S. patent number), pharmaceutical and secretable peptides (U.S. patent number, 6,140,075, and number), improved processing traits (U.S. patent number), improved digestibility (U.S. patent number), low raffinose (U.S. patent number), industrial enzyme production (U.S. patent number), improved taste (U.S. patent number), nitrogen fixation (U.S. patent number), hybrid seed production (U.S. patent number 5,689,041), fiber yield (U.S. patent number; number, and number), and biofuel production (U.S. patent number).

Alternatively, agronomically advantageous genes or transgenes may affect the above plant characteristics or phenotypes by encoding RNA molecules that cause targeted modulation of gene expression of endogenous genes, for example by antisense (see, e.g., U.S. Pat. No. 5,107,065); inhibitory RNAs ("RNAi", including regulation of gene expression by miRNA-, siRNA-, trans-acting siRNA-, and staged sRNA-mediated mechanisms, e.g., as described in published applications u.s.2006/0200878 and u.s.2008/0066206, and U.S. patent application 11/974,469); or co-suppression mediated mechanisms. The RNA may also be a catalytic RNA molecule (e.g., a ribozyme or riboswitch; see, e.g., US 2006/0200878) engineered to cleave a desired endogenous mRNA product. Methods for constructing constructs and introducing them into cells in such a way that a transcribable DNA sequence is transcribed into an RNA molecule capable of causing gene suppression are known in the art.

Selectable markers

Selectable marker transgenes may also be used with the regulatory elements of the present invention. As used herein, the term "selectable marker transgene" refers to any transcribable DNA sequence whose expression or lack of expression in a transgenic plant, tissue or cell can be screened or scored in some manner. Selectable marker genes and their associated selection and screening techniques for practicing the present invention are known in the art and include, but are not limited to, transcribable DNA sequences encoding β -Glucuronidase (GUS), green Fluorescent Protein (GFP), proteins conferring antibiotic resistance, and proteins conferring herbicide resistance. Examples of selectable marker transgenes are provided as GOI-At.ShkG-CTP2+AGRtu. AroA-CP4.Nat:1 (SEQ ID NOs: 47) for selection of transformed plant cells by glyphosate selection, GOI-At.ShkG-CTP2+ec.aadA-SPC/STR:1:1 (SEQ ID NO: 54) for selection of transformed plant cells by spectinomycin selection, and GOI-ec.ui dA+St.LS1:3 (SEQ ID NO: 50) for the GUS reporter gene in the transgene expression cassette in the examples below, which are intended to be retained in the integration construct after automatic excision to demonstrate retention of the cassette and to determine zygosity.

Site-specific nucleases

As used herein, the term "genome editing" refers to modifying the genetic sequence of a target site in a DNA molecule or genome or chromosome of a living organism or cell (such as the genome of an agricultural crop) by deletion, substitution, and/or insertion of the DNA sequence at or near the target site (which may be generated using a site-specific nuclease). "site-specific integration" or "site-directed integration" refers to the term of inserting a DNA sequence or construct into the genome or chromosome of a living organism or cell at a target site. As used herein, the term "site-specific nuclease" refers to a DNA cleaving nuclease that produces a double strand break or nick at or near a particular target site or position of a DNA molecule, chromosome or genome. As used herein, a "target site" for genome editing refers to a location of a polynucleotide sequence within a plant genome that is bound and cleaved by a site-specific nuclease, thereby introducing a double-strand break (or single-strand nick) into the nucleic acid backbone of the polynucleotide sequence and/or its complementary DNA strand. After making the break or cleavage, the DNA repair mechanism of the cell can recognize and repair the break or cleavage via non-homologous end joining (NHEJ) or homology directed repair, and possibly introduce mutations and/or insertions at the target site, as understood in the art.

The site-specific nucleases provided herein may be selected from the group consisting of: zinc Finger Nucleases (ZFNs), meganucleases, RNA-guided endonucleases, such as CRISPR-associated nucleases, TALE endonucleases (TALENs), recombinases, transposases or any other endonuclease that is likely. See, e.g., khandagaale, K.et al, "Genome editing for targeted improvement in plants," Plant Biotechnol Rep 10:327-343 (2016); and Gaj, t et al, "ZFN, TALEN and CRISPR/Cas-based methodsfor genome engineering," Trends biotechnol.31 (7): 397-405 (2013), the contents and disclosure of which are incorporated herein by reference. The expression cassettes provided herein may encode a site-specific nuclease. Such an expression cassette may comprise a transcribable DNA sequence encoding a site-specific nuclease operably linked to a plant-expressible promoter. In another aspect, a recombinant DNA construct provided herein may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten expression cassettes encoding one or more site-specific nucleases.

According to embodiments of the present disclosure, the recombinase may be a serine recombinase linked to a DNA recognition motif, a tyrosine recombinase linked to a DNA recognition motif, or other recombinases known in the art. The recombinase or transposase may be a DNA transposase or a recombinase linked to a DNA binding domain. The tyrosine recombinase linked to the DNA recognition motif may be selected from the group consisting of: cre recombinase, flp recombinase and Tnp 1 recombinase. According to some embodiments, the Cre recombinase or Gin recombinase provided herein is tethered to a zinc finger DNA binding domain. In another embodiment, the serine recombinases provided herein linked to a DNA recognition motif are selected from the group consisting of: phiC31 integrase, R4 integrase and TP-901 integrase. In another embodiment, the DNA transposase provided herein that is linked to a DNA binding domain is selected from the group consisting of TALE-piggyBac and TALE-multiplexer.

According to embodiments of the present disclosure, the RNA guided endonuclease or CRISPR-associated nuclease may be selected from the group consisting of: non-limiting examples of Cas1, cas1B, cas2, cas3, cas4, cas5, cas6, cas7, cas8, cas9 (also known as Csn1 and Csx 12), cas10, csy1, csy2, csy3, cse1, cse2, csc1, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmr1, cmr3, cmr4, cmr5, cmr6, csb1, csb2, csb3, csx17, csx14, csx10, csx16, csaX, csx3, csx1, csx15, csf1, csf2, csf3, csf4, cpf1, casX, casY and their homologs or modified forms, argonaute (Argonaute proteins) include thermophilic bacteria (84) Argonaute (TtAgo), geobacillus (Pyrococcus furiosus) and other variants of the same, or other saline-alkaline and alkaline variants (according to the mangostin) of the mangrover 35, in another aspect, the site-specific nucleases provided herein are selected from the group consisting of Cas9 or Cpf1 enzymes.

For RNA-guided endonucleases or CRISPR-associated nucleases, a guide RNA (gRNA) molecule may be required to direct the endonuclease to a target site in a DNA molecule, chromosome or genome of a plant by base pairing or hybridization to cause DSBs or nicks at or near the target site. The gRNA may be transformed or introduced into a plant cell or tissue as a recombinant DNA construct (possibly together with a nuclease or a DNA construct encoding a nuclease) comprising a transcribable DNA sequence encoding a guide RNA operably linked to a plant-expressible promoter. As understood in the art, a "guide RNA" may include, for example, CRISPR RNA (crRNA), single stranded guide RNA (sgRNA) or any other RNA molecule that can direct or direct an endonuclease to a particular target site in the genome. A "single stranded guide RNA" (or "sgRNA") is an RNA molecule comprising crRNA covalently linked to tracrRNA via a linker sequence, which can be expressed as a single RNA transcript or molecule. The guide RNA comprises a guide or targeting sequence that is identical or complementary to a target site within a DNA molecule, chromosome, or plant genome. Protospacer Adjacent Motifs (PAMs) may be present in the genome immediately 5 'to and upstream of the genomic target site sequence complementary to the targeting sequence of the guide RNA-i.e., immediately downstream (3') of the sense (+) strand of the genomic target site (relative to the targeting sequence of the guide RNA), as is known in the art. See, e.g., wu, x. Et al, "Target specificity of the CRISPR-Cas9 system," Quant biol.2 (2): 59-70 (2014), the contents and disclosure of which are incorporated herein by reference. Genomic PAM sequences on the sense (+) strand adjacent to the target site (relative to the targeting sequence of the guide RNA) may comprise 5'-NGG-3'. However, the corresponding sequence of the guide RNA (i.e., immediately downstream (3') of the targeting sequence of the guide RNA) may not generally be complementary to the genomic PAM sequence. The guide RNA may typically be a non-coding RNA molecule that does not code for a protein. The guide sequence of the guide RNA can be at least 10 nucleotides in length, such as 12-40 nucleotides, 12-30 nucleotides, 12-20 nucleotides, 12-35 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, or 17-25 nucleotides in length, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length. The guide sequence may have at least 95%, at least 96%, at least 97%, at least 99% or 100% identity or complementarity to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more consecutive nucleotides of the DNA sequence at the target site. The expression cassettes provided herein can encode guide RNAs. Such an expression cassette may comprise a transcribable DNA sequence encoding a guide RNA operably linked to a plant-expressible promoter. In another aspect, a recombinant DNA construct provided herein may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten expression cassettes encoding one or more guide RNAs.

Zinc Finger Nucleases (ZFNs) are synthetic proteins consisting of an engineered zinc finger DNA binding domain fused to a cleavage domain (or cleavage half-domain), which can be derived from a restriction endonuclease (e.g., fok 1). The DNA binding domain may be canonical (C2H 2) or atypical (e.g., C3H or C4). Depending on the target site, the DNA binding domain may comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more zinc fingers). Multiple zinc fingers in a DNA binding domain may be separated by one or more linker sequences. ZFNs can be designed to cleave almost any segment of double stranded DNA by modification of the zinc finger DNA binding domain. ZFNs form dimers from monomers that include a non-specific DNA cleavage domain (e.g., derived from fokl nuclease) fused to a DNA binding domain that includes a zinc finger array engineered to bind to a target site DNA sequence. The DNA-binding domain of ZFNs can typically consist of 3-4 (or more) zinc fingers. Amino acids at the-1, +2, +3, and +6 positions relative to the start of the zinc finger alpha-helix that promotes site-specific binding to the target site can be varied and tailored to suit the particular target sequence. Other amino acids may form a consensus backbone to generate ZFNs with different sequence specificities. Methods and rules for designing ZFNs that target and bind to specific target sequences are known in the art. See, for example, U.S. patent application nos. 2005/0064474, 2009/011717, and 2012/0142062, the contents and disclosures of which are incorporated herein by reference. The Fok1 nuclease domain may need to dimerize in order to cleave DNA, and thus two ZFNs with their C-terminal regions are required to bind opposite DNA strands (5-7 bp apart) of the cleavage site. If the double ZF binding site is palindromic, ZFN monomers can cleave the target site. As used herein, ZFNs are broad and include monomeric ZFNs that can cleave double-stranded DNA without the aid of another ZFN. The term ZFN may also be used to refer to one or both members of a pair of ZFNs engineered to co-act to cleave DNA at the same site.

Without being limited by any scientific theory, because the DNA binding specificity of the zinc finger domain can be re-engineered using one of a variety of methods, custom ZFNs can in theory be constructed to target virtually any target sequence (e.g., at or near the GA oxidase gene in the plant genome). Publicly available methods for engineering zinc finger domains include Context-dependent assembly (Context-dependent Assembly (CoDA)), oligomerization pool engineering (Oligomerized Pool Engineering (OPEN)), and modular assembly. In one aspect, the methods and/or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more ZFNs. In another aspect, ZFNs provided herein are capable of generating targeted DSBs or nicks. In one aspect, a vector comprising a polynucleotide encoding one or more, two or more, three or more, four or more, or five or more ZFNs is provided to a cell by transformation methods known in the art (e.g., without limitation, viral transfection, particle bombardment, PEG-mediated protoplast transfection, or agrobacterium-mediated transformation). ZFNs can be introduced as ZFN proteins, as polynucleotides encoding ZFN proteins, and/or as a combination of proteins and polynucleotides encoding proteins.

Meganucleases, such as the LAGLIDADG family of homing endonucleases, typically identified in microorganisms, are unique enzymes with high activity and long recognition sequences (> 14 bp) that lead to site-specific digestion of target DNA. Engineered forms of naturally occurring meganucleases typically have an extended DNA recognition sequence (e.g., 14 to 40 bp). According to some embodiments, the meganuclease may comprise a scaffold or base enzyme selected from the group consisting of: I-CreI, I-CeuI, I-MsoI, I-SeeI, I-AniI and I-Dmo I. The engineering of meganucleases can be more challenging than ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are interleaved together in a single domain. Specific mutagenesis and high throughput screening methods have been used to create novel meganuclease variants that recognize unique sequences and have improved nuclease activity. Thus, meganucleases can be selected or engineered to bind genomic target sequences in plants. In one aspect, the methods and/or compositions provided herein comprise one or more, two or more, three or more, four or more, or five or more meganucleases. In another aspect, meganucleases can generate targeted nicks or breaks.

Zinc Finger Nucleases (ZFNs) and TAL effector nucleases (TALENs) are chimeric enzymes that combine nucleases and DNA binding domains. TALENs are a class of sequence-specific nucleases that can be used to create double strand breaks at specific target sequences in the genome of a plant or other organism. TALENs are restriction enzymes produced by fusing a transcription activator-like effector (TALE) DNA binding domain with a nuclease domain (e.g., fokl). When each member of the TALEN pair binds to a DNA site flanking the target site, the fokl monomers dimerize and cause double-stranded DNA breaks at the target site. In addition to the wild-type fokl cleavage domain, variants with mutated fokl cleavage domains have been designed to improve cleavage specificity and cleavage activity. The fokl domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with the proper orientation and spacing. The number of amino acid residues between the TALEN DNA binding domain and the fokl cleavage domain, as well as the number of bases between two separate TALEN binding sites, are parameters to achieve high activity levels. TALENs are artificial restriction enzymes produced by fusing a transcription activator-like effector (TALE) DNA binding domain to a nuclease domain. In some aspects, the nuclease is selected from the group consisting of: pvuII, mutH, tevI, fokI, alwI, mlyI, sbfI, sdaI, stsI, cleDORF, clo051 and Pept071. When each member of the TALEN pair binds to a DNA site flanking the target site, the fokl monomers dimerize and cause double-stranded DNA breaks at the target site. As used herein, the term TALEN is broad and includes monomeric TALENs that can cleave double-stranded DNA without the aid of another TALEN. The term TALEN also refers to one or both members of a pair of TALENs that co-act at the same site to cleave DNA.

Transcription activator-like effectors (TALEs) can be engineered to bind virtually any DNA sequence at or near the genomic locus of a GA oxidase gene, such as in a plant. TALE has a central DNA binding domain consisting of 13-28 repeat monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved except for the hypervariable amino acid residues at positions 12 and 13. The DNA binding domain of TAL effectors may contain 33-35 amino acid sequence repeats, including repeated variable double Residues (RVDs) at residues 12 and 13, which determine their specificity in DNA binding. Each repeat binds to a specific nucleotide that facilitates engineering of a specific DNA binding domain by selecting a combination of repeat segments containing the appropriate RVDs. The number of repeats of the RVD sequence determines the length and sequence of the target sequence to be identified (Podevin et al, (2013) Trends in Biotechnology31 (6): 375-383). Two variable amino acids are referred to as repeated variable double Residues (RVD). Amino acid pairs of RVDs preferentially recognize certain nucleotide bases, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition allows for engineering of a particular DNA binding domain by selecting a combination of repeat segments containing the appropriate RVDs.

In addition to the wild-type fokl cleavage domain, variants with mutated fokl cleavage domains have been designed to improve cleavage specificity and cleavage activity. The fokl domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with the proper orientation and spacing. The number of amino acid residues between the TALEN DNA binding domain and the fokl cleavage domain, as well as the number of bases between two separate TALEN binding sites, are parameters to achieve high activity levels. PvuII MutH and TevI cleavage domains are useful alternatives to FokI and FokI variants for use with TALE. When coupled to TALE, pvuII functions as a highly specific cleavage domain (see Yank et al 2013.PLoS One.8:e82539). MutH is capable of introducing strand-specific nicks in DNA (see Gabsalilow et al, 2013.Nucleic Acids Research.41:e83). TevI introduces a double strand break in DNA at the targeting site (see beudeley et al, 2013.Nature Comm.4:1762). The relationship between the amino acid sequence and DNA recognition of the TALE binding domain allows for a protein to be designed. The TALE construct may be designed using a software program such as DNA Works. Other methods of designing TALE constructs are known to those skilled in the art. See Doyle et al, nucleic Acids Research (2012) 40:wl 17-122; cermak et al, nucleic Acids Research (2011) 39:e82; and tale-nt.cac.comell.edu/about. In one aspect, the recombinant DNA constructs provided herein comprise one or more, two or more, three or more, four or more, or five or more TALENs. In another aspect, a TALEN provided herein is capable of generating a targeted nick or break at a target site.

A Zinc Finger Nuclease (ZFN) comprises a zinc finger DNA binding domain and a double cleavage inducing domain. Recognition site specificity is conferred by zinc finger domains, which may contain two, three, or four zinc fingers, e.g., having a C2H2 structure, although other zinc finger structures are known and have been engineered. The zinc finger domain is suitable for use in designing polypeptides that specifically bind to selected polynucleotide recognition sequences. ZFNs consist of engineered DNA-binding zinc finger domains linked to non-specific endonuclease domains (e.g., nuclease domains from type IIs endonucleases, such as fokl). Other functionalities may be fused to the zinc finger binding domain, including transcriptional activator domains, transcriptional repressor domains, and methylases. In some examples, dimerization of nuclease domains is essential for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, the three-finger domain recognizes a sequence of nine consecutive nucleotides with dimerization requirements of nucleases, using two sets of zinc finger triplets to bind to an eighteen nucleotide recognition sequence (Gaj et al, (2013) Trends Biotechnology,31 (7): 397-405; and Urnov et al, (2010) Nature Reviews Genetics,11: 636-646).

Cell transformation

The invention also relates to a method of producing transformed cells and plants comprising one or more regulatory elements operably linked to a transcribable DNA sequence.

The term "transformation" refers to the introduction of a DNA molecule into a recipient host. As used herein, the term "host" refers to a bacterium, fungus, or plant, including any cell, tissue, organ, or progeny of a bacterium, fungus, or plant. Plant tissues and cells of particular interest include protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos and pollen.

As used herein, the term "transformed" refers to a cell, tissue, organ or organism into which a foreign DNA molecule (such as a construct) has been introduced. The introduced DNA molecule may be integrated into the genomic DNA of a recipient cell, tissue, organ or organism such that the introduced DNA molecule is inherited by subsequent offspring. "transgenic" or "transformed" cells or organisms may also include progeny of a cell or organism, as well as progeny resulting from a breeding program that uses such transgenic organisms as parents for hybridization and that exhibits an altered phenotype due to the presence of foreign DNA. The introduced DNA molecule may also be transiently introduced into the recipient cell such that the introduced DNA molecule is not inherited by subsequent offspring. The term "transgenic" refers to a bacterium, fungus, or plant that contains one or more heterologous DNA molecules.

There are many methods known to those skilled in the art for introducing DNA molecules into plant cells and transforming plant cells. The process generally includes the steps of: selecting a suitable host cell or explant, transforming said cell or explant with a molecule or vector, and obtaining a transformed cell. In the practice of the present invention, the methods and materials for transforming plant cells by introducing plant constructs into the plant genome may include any well known and proven methods. Suitable methods include, but are not limited to, bacterial infection (e.g., agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., through PEG-mediated transformation, drying/inhibition-mediated DNA uptake, electroporation, agitation by silicon carbide fibers and acceleration by DNA coated particles), gene editing (e.g., CRISPR-Cas system), and the like. According to certain embodiments, the transformation method comprises agrobacterium-or rhizobium-mediated transformation or particle bombardment or particle-mediated transformation.

The host cell may be any cell or organism, such as a plant cell, an algal cell, an algae, a fungal cell, a fungus, a bacterial cell or an insect cell. In particular embodiments, host cells and transformed cells may include cells from crop plants.

Transgenic plants can then be regenerated from the transgenic plant cells of the invention. Seeds may be produced from such transgenic plants using conventional breeding techniques or self-pollination. Such seeds and progeny plants grown from such seeds will contain the recombinant DNA molecules of the invention and will therefore be transgenic.

The transgenic plants of the invention may be self-pollinated to provide seeds for homozygous transgenic plants of the invention (homozygous for the recombinant DNA molecule) or may be crossed with non-transgenic plants or different transgenic plants to provide seeds for heterozygous transgenic plants of the invention (heterozygous for the recombinant DNA molecule). Such homozygous and heterozygous transgenic plants are referred to herein as "progeny plants". The progeny plant is a transgenic plant that is inherited from the original transgenic plant and contains the recombinant DNA molecules of the invention. Seeds produced using the transgenic plants of the invention can be harvested and used to culture the generations of the transgenic plants, i.e., the progeny plants of the invention, which comprise the constructs of the invention and express genes of agronomic interest. Descriptions of breeding methods commonly used for different crops can be found in one of several references, see, for example, al lard, principles of Plant Breeding, john Wiley & Sons, NY, u.of CA, davis, CA,50-98 (1960); simmonds, principles of Crop Improveme nt, longman, inc, NY,369-399 (1979); sneep and Hendriksen, plant breeding Perspectives, wageningen (edit), center for Agricultural Publis hing and Documentation (1979); fehr, soybeans: improvement, product ion and Uses, 2 nd edition, monograph,16:249 (1987); fehr, principles of Variety Development, theory and Technique, (volume 1) and Crop Species Soybean (volume 2), iowa State uni, macmillan pub.co., NY,360-376 (1987).

The transformed plant may be analyzed for the presence of the gene or genes of interest and the level and/or profile of expression conferred by the regulatory elements of the invention. Those skilled in the art will be aware of numerous methods that can be used to analyze transformed plants. For example, methods for plant analysis include, but are not limited to, southern or Northern blotting, PCR-based methods, biochemical analysis, phenotypic screening methods, field evaluation, and immunodiagnostic assays. Can be used as described by the manufacturer(Applied Biosystems, foster City, calif.) reagents and methods to measure expression of transcribable DNA sequences and use +.>The Testing Matrix determines the number of PCR cycles. Alternatively, +.>(Third Wave Technologies, madison, wis.) reagents and methods to assess transgene expression.

The invention also provides parts of the plants of the invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. Plant parts of the invention may be viable, non-viable, renewable and/or non-renewable. The invention also includes and provides transformed plant cells comprising the DNA molecules of the invention. Transformed or transgenic plant cells of the invention include regenerable and/or non-regenerable plant cells.

The invention also provides commercial products produced from transgenic plants or parts thereof comprising the recombinant DNA molecules of the invention. The commercial product of the present invention contains a detectable amount of DNA comprising a DNA sequence selected from the group consisting of: SEQ ID NOS 1-26, 59-62 and 64-66. As used herein, "commodity product" refers to any composition or product that consists of material derived from a transgenic plant, seed, plant cell, or plant part that contains a recombinant DNA molecule of the invention. Commercial products include, but are not limited to, processed seeds, kernels, plant parts, and meal. The commercial product of the present invention will contain a detectable amount of DNA corresponding to the recombinant DNA molecule of the present invention. Detection of one or more such DNA in a sample may be used to determine the content or source of a commodity product. Any standard method of DNA molecule detection may be used, including the detection methods disclosed herein.

The invention may be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention unless otherwise indicated. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, and therefore all matter set forth is to be interpreted as illustrative and not in a limiting sense.

Examples

Example 1

Identification of regulatory elements capable of driving automatic excision in crop plants

This example provides regulatory elements identified through years of experimentation that can drive efficient automatic excision in transgenic corn, soybean and cotton.

By combining literature searches with public and proprietary database searches, regulatory elements that are likely to drive efficient automatic excision in transgenic crop plants were first identified. Effective automatic excision has been determined using the Cre/Lox recombinase system on seventy soybean, twenty cotton and one hundred maize binary transformation vector constructs containing different regulatory elements and combinations. From these studies, a small number of regulatory elements that provide effective automatic excision were identified and presented in table 1 below.

Table 1. Regulatory elements providing efficient automatic excision in crop plants.

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Example 2

Automatic excision in maize and Rice CDC45-1 promoters driven stably transformed maize plants

Maize plants are transformed with recombinant DNA molecules, particularly plant transformation constructs comprising different regulatory elements that drive Cre-recombinase expression, to assess the ability and efficiency of automatic excision of Cre-recombinase driven Cre-recombinase expression cassettes and marker gene expression cassettes expressed under the control of the different regulatory elements.

Transformation of maize plants with binary plant transformation constructs comprising the following three transgene expression cassettes: a selectable marker gene expression cassette and a Cre-recombinase expression cassette, both flanked by two LoxP sites (RS-P1. Lox1:1, SEQ ID NO: 44), and a third expression cassette located outside the LoxP sites, which expresses a beta-Glucuronidase (GUS) transgene. The Cre-recombinase expression cassettes were used to determine the different EXPs to test their ability to drive efficient automatic excision of the Cre-recombinase expression cassette and the marker gene expression cassette located between LoxP sites. The Cre-recombinase expression cassette consists of the following elements: the EXP to be tested is operably linked to 5 'of a synthetic coding sequence encoding Cre-recombinase (e.g., a codon redesigned for expression in plant cells) (GOI-P1. Cre-St. LS1:1, SEQ ID NO: 43) containing a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (GenBank accession number: X04753) operably linked to 5' of a 3 'termination or 3' UTR (T-AGRtu. Nos:13, SEQ ID NO: 48). Each plant transformation construct contained one of two different marker gene expression cassettes, designated "marker-1" and "marker-2". The marker-1 marker gene expression cassette comprises the following elements: constitutive EXP (EXP-Os. Act1:1:1 (SEQ ID NO: 46)), operably linked to 5' of a synthetic coding sequence encoding a plastid-targeted CP4 coding sequence (GOI-At. ShkG-CTP2+AGRtu. AroA-CP4.Nat:1, SEQ ID NO: 47) that provides tolerance to the herbicide glyphosate, operably linked to 5' of a 3' termination region T-AGRtu. Nos. 13 (SEQ ID NO: 48). The marker-2 gene expression cassette comprises the following elements: constitutive EXP (EXP-Os. TubA-3:1 (SEQ ID NO: 52)), operably linked to 5 'of a synthetic coding sequence encoding a plastid-targeted CP4 coding sequence (GOI-At. ShkG-CTP2+AGRtu. AroA-CP4.Nat:1, SEQ ID NO: 47) that provides tolerance to the herbicide glyphosate, operably linked to a 3' termination region

T-AGRtu. Nos. 13 (SEQ ID NO: 48).

The marker gene cassette and the Cre-recombinase cassette are flanked by two loxP Cre-recombinase recognition sequences (RS-P1. Lox1:1, SEQ ID NO: 44) in the same head-to-tail direction. If automatic excision is effective, expression of Cre-recombinase in the transformed plant cell is expected to result in excision of both cassettes. The GUS expression cassette was cloned outside the LoxP Cre-recombinase recognition sequence and contains the following elements: EXP (EXP-Os.Act1+CaMV.35S.2xA1-B3+Ta.Lhcb1:1:1, SEQ ID NO: 49) comprising chimeric promoter and leader sequence is operably linked to 5' of a synthetic coding sequence encoding β -glucuronidase (GOI-ec.uidA+St.LS1:3, SEQ ID NO: 50) containing a processable intron derived from potato light-induced tissue specific ST-LS1 gene (GenBank accession number: X04753) operably linked to 5' of a 3' termination region (T-St.Pis 4-1:4:1,SEQ ID NO:51).

Three EXPs derived from corn and rice CDC45 gene homologs were operably linked to a transcribable DNA sequence encoding Cre recombinase and their ability to drive automatic excision in stably transformed corn plants was determined. EXP-Zm.Cdc45-1+Zm.DnaK:1:1 (SEQ ID NO: 1) comprises a promoter (P-Zm.Cdc45-1:8, SEQ ID NO: 2) operably linked to 5 'of a synthetic leader sequence (L-Zm.Cdc45-1:1, SEQ ID NO: 3) operably linked to 5' of an intron (I-Zm.DnaK: 1, SEQ ID NO: 39). EX P-Os.Cdc45-1:1:1 (SEQ ID NO: 4) comprises a promoter (P-Os.Cdc45-1-1:1:1,SEQ ID NO:5) operably linked to 5' of the leader sequence (L-Os.Cdc45-1-1:1:1,SEQ ID NO:6). EXP-Zm.Cdc45-2+Zm.DnaK:1:2 (SEQ ID NO: 27) comprises a promoter (P-Zm.Cdc45-2-1:1:3,SEQ ID NO:28) operably linked to 5 'of a leader sequence (L-Zm.Cdc45-2-1:1:1,SEQ ID NO:29) operably linked to 5' of an intron (I-Zm.DnaK: 1, SEQ ID NO: 39).

Two additional EXPs were also operably linked to a transcribable DN a sequence encoding Cre recombinase and their ability to drive automatic excision in stably transformed maize plants was determined. EXP-Zm.Zm13:2 (SEQ ID NO: 30) from the germline preference gene expressed in pollen comprises a promoter (P-Zm.Zm13:2, SEQ ID NO: 31) operably linked to 5' of the leader sequence (L-Zm.Zm13:2, SEQ ID NO: 32). EXP-Zm.wall+Zm.DnaK from endosperm preference gene 1:5 (SEQ ID NO: 33) comprises a promoter (P-Zm.wall-1:1:9,SEQ ID NO:34) operably linked to 5 'of a leader sequence (L-Zm.wall-1:1:1,SEQ ID NO:35) operably linked to 5' of an intron (I-Zm.DnaK: 1, SEQ ID NO: 39).

Transformation of maize plant cells by agrobacterium-mediated transformation using the binary plant transformation construct described above, wherein each of the EXPs described above to be tested is operably linked to a transcribable DNA sequence encoding a Cre recombinase. Methods for agrobacterium-mediated transformation are well known in the art. The resulting transformed plant cells are regenerated into maize plants under glyphosate selection.

Selecting R with single copy event ₀ Plants and allowed to self-pollinate. Then use TAQMAnalysis of the obtained R by assay ₁ The presence of Cre, CP4 and GUS transgenes in seeds. Also using GUS transgene +.>Determination of R by assay ₁ Zygosity of seeds for the integration construct. According to each selected R ₀ Event selfing, forty-one R was determined ₁ Seed. Those producing at least two R's that are homozygous for GUS and lack Cre and CP4 transgenes ₁ The event of the seed is believed to successfully automatically cut off the Cre and CP4 transgene expression cassettes. Table 2 below shows the results for each EXP. Data corresponding to two different CP4 marker transgene cassettes (marker-1 and marker-2) are also presented in table 2. Only one of the CP4 transgene cassettes was used for EXP-Zm.Zm13:2 (marker-1) and EXP-Zm.wall+Zm.DnaK:1:5 (marker-2). In Table 2, the column "percent of homozygous event/analyzed 41 seeds" indicates the production of at least two R ₁ Percentage of events for seeds that are homozygous for GUS and lack Cre and CP4 transgenes. In brackets, the first digit indicates that at least two R particles are produced ₁ R of seed ₀ Number of events, the seed was homozygous for GUS and lacks the Cre and CP4 transgenes, the second number represents R where each event analyzed 41 seeds ₀ Number of events.

TABLE 2 demonstration of efficient autoproteological R in stably transformed maize plants with maize and Rice CDC45-1 promoter driven autoproteological excision ₀ Percentage of events.

As can be seen from Table 2 above, the promoters contained within EXP-Zm.Cdc45-1+Zm.DnaK:1:1 (P-Zm.Cdc45-1:8, SEQ ID NO: 2) and EXP-Os.Cdc45-1:1:1 (P-Os.Cdc45-1-1:1:1,SEQ ID NO:5) were very effective in driving automatic excision in stably transformed maize plants. Promoters contained within EXP-zm.Cdc45-2+zm.DnaK:1:2 (P-zm.Cdc45-2-1:1:3,SEQ ID NO:28), EXP-zm.zm13:2 (P-zm.zm13:2, SEQ ID NO: 31) and EXP-zm.wall+zm.DnaK:1:5 (P-zm.wall-1:1:9,SEQ ID NO:34) were unable to drive automatic excision in stably transformed maize plants in this experiment.

Example 3

Arabidopsis CDC45 promoter and other germ line preferred promoters drive automatic excision in stably transformed soybean plants

Soybean plants were transformed with recombinant DNA molecules, particularly plant transformation constructs comprising different regulatory elements that drive Cre-recombinase expression, to assess the ability and efficiency of Cre-recombinase expression cassettes and marker gene expression cassettes expressed under the control of the different regulatory elements to automatically excise.

Transforming a soybean plant with a binary plant transformation construct comprising the following four transgene expression cassettes: generating a selectable marker gene expression cassette (crtB) for the color phenotype; cre-recombinase expression cassette and a selectable marker expression cassette (aadA) for selecting transformed soybean cells flanked by two loxP sites (RS-P1. Lox. TATA-R9-1:1:1,SEQ ID NO:45); and a fourth expression cassette located outside the LoxP site, which expresses a β -Glucuronidase (GUS) transgene. The Cre-recombinase expression cassettes were used to determine the different EXPs to test their ability to drive efficient automatic excision of crtB, cre-recombinase and marker gene expression cassettes all located between LoxP sites. The Cre-recombinase expression cassette consists of the following elements: EXP to be tested, operatively linked to 5' of a synthetic coding sequence encoding Cre-recombinase (GOI-P1. Cre-St. LS1:1:1, SEQ ID NO: 43) containing a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (GenBank accession number: X04753), operatively linked to 5' of a 3' termination region (T-AGRtu. Nos:13, SEQ ID NO: 48). The color marker gene expression cassette comprises a seed-preferred promoter and leader sequence (P-Br. Snap2-1:1:20,SEQ ID NO:56) operably linked to 5' of a synthetic coding sequence encoding a chloroplast-targeted (TS-Ps. RbcS-3C-1:3:1,SEQ ID NO:57) phytoene synthase (crtB) (CR-PANag. CrtB. Nno-1:4:1,SEQ ID NO:58) operably linked to 5' of a 3' termination region (T-Br. Snap2-1:3:6,SEQ ID NO:59). Expression of the crtB gene in seeds produced orange colored seeds and was an indication that the transgene cassette remained between LoxP sites due to failure to excise the cassette after automatic excision. The transformation aadA selection marker cassette consists of the following elements: EXP (EXP-at. Act7:2, SEQ ID NO: 53) operably linked to 5' encoding a chloroplast-targeted Tn7 adenylyl transferase coding sequence (GOI-at. ShkG-CTP2+ec. AadA-SPC/STR:1:1, SEQ ID NO: 54) conferring resistance to spectinomycin and used to select transformed plant cells operably linked to 5' of a 3' termination region (T-AGRtu. Nos:13, SEQ ID NO: 48). The GUS transgene expression cassette comprises an enhanced cauliflower mosaic virus 35S promoter and leader sequence (EXP-CaMV. 35S-enh:1:2, SEQ ID NO: 55) operably linked to 5' of a synthetic coding sequence encoding β -glucuronidase (GOI-ec. UidA+St. LS1:3, SEQ ID NO: 50) containing a processable intron derived from the potato light-induced tissue specific ST-LS1 gene (GenBank accession number: X04753), operably linked to 5' of a 3' termination region (T-AGRtu. Nos:13, SEQ ID NO: 48).

Seven EXPs derived from arabidopsis genes with germ line preferred expression were operably linked to a transcribable DNA sequence encoding Cre recombinase and their ability to drive automatic excision in stably transformed soybean plants was determined. See table 3 below. Each Cre-recombinase expression cassette also comprises a native 3' termination region (also presented in Table 3) corresponding to the gene from which the EXP is derived. Two of EXPs, EXP-at.Swi1 (SEQ ID NO: 13) and EXP-at.Swi1s (SEQ ID NO: 14), are variants of different lengths derived from the same germline bias gene and comprise a promoter operably linked to a leader sequence. The 3' UTR, i.e.T-At.Swi1-1:2:1 (SEQ ID NO: 14) was used in both EXP-At.Swi1 expression cassettes. Likewise, the other two EXPs, EXP-Syn1 (SEQ ID NO: 36) and EXP-Syn1a (SEQ ID NO: 37), are variants of different lengths derived from the same germline bias gene and comprise a promoter operably linked to a leader sequence. The 3' UTR, i.e.T-At.Syn1-1:2:1 (SEQ ID NO: 38) was used in both EXP-At.Syn1 expression cassettes.

Transformation of soybean plant cells by agrobacterium-mediated transformation using the binary plant transformation construct described above, wherein each of the EXPs described above to be tested is operably linked to a transcribable DNA sequence encoding a Cre recombinase. Methods for agrobacterium-mediated transformation are well known in the art. The resulting transformed plant cells are regenerated into soybean plants under selection of spectinomycin.

Selecting R with single copy event ₀ Plants and allowed to self-pollinate. And then useAnalysis of the obtained R by assay ₁ Presence of GUS transgene in seeds. Successful auto excision of Cre, aadA and crtB expression cassettes between LoxP sites was deduced from the lack of orange color conferred to seeds by the retained crtB expression cassette. Table 3 below shows the results for each EXP/3' UTR combination.

TABLE 3 production of marker-free R in stably transformed soybean plants ₁ R of seed ₀ Percentage of events.

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As can be seen from Table 3 above, in this experimentAll EXPs (except EXP-Syn1 and EXP-Syn1 a) were able to drive automatic excision in stably transformed soybean plants. The combination of EXP and 3' UTR derived from the Arabidopsis CDC45 gene (EXP-at. Cdc45:1:1, SEQ ID NO:9 and T-at. Cdc45:1, SEQ ID NO: 12) provided the highest percentage of production of marker-free R ₁ R of seed ₀ An event. Two variants EXP derived from the Arabidopsis Swi1 gene (EXP-At.Swi1, SEQ ID NO:13 and EXP-At.Swi1a, SEQ ID NO: 14) and the same corresponding 3' UTR (T-At.Swi1-1:2:1,SEQ ID NO:15) also provided a high percentage of the generation of marker-free R ₁ R of seed ₀ An event.

Example 4

Soybean Rsp-1 promoter drives automatic excision in stably transformed soybean plants

Soybean plants are transformed with constructs, particularly plant binary transformation constructs comprising test regulatory elements that drive Cre-recombinase expression, and used to assess the ability and efficiency of automatic excision of Cre-recombinase expression cassettes and marker gene expression cassettes.

Transforming a soybean plant with a binary plant transformation construct comprising the following three transgene expression cassettes: cre-recombinase expression cassette and a selectable marker expression cassette (aadA) for selecting transformed soybean cells flanked by two loxP sites (RS-P1. Lox1:1, SEQ ID NO: 44); and a third expression cassette outside the LoxP site for expressing a beta-Glucuronidase (GUS) transgene. Cre-recombinase expression cassettes are used to determine their ability of different EXPs to drive efficient automatic excision of Cre-recombinase expression cassettes and marker gene expression cassettes. The Cre-recombinase expression cassette consists of the following elements: test EXP operably linked to 5' of a synthetic coding sequence encoding Cre-recombinase (GOI-P1. Cre-St. LS1:1:1, SEQ ID NO: 43) containing a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (GenBank accession number X04753) operably linked to 5' of a 3' termination region (T-At. Cd c45:1, SEQ ID NO: 12). The transformation selection marker cassette aadA consists of the following elements: EXP (EXP-at. Act7:2, SEQ ID NO: 54) operably linked to 5' encoding a chloroplast-targeted Tn7 adenylyl transferase coding sequence (GOI-at. ShkG-CTP2+ec. AadA-SPC/STR:1:1, SEQ ID NO: 54) that confers resistance to spectinomycin and is used to select transformed plant cells, said coding sequence being operably linked to 5' of a 3' termination region (T-AGRtu. Nos. 13, SEQ ID NO: 48). The GUS transgene expression cassette consists of the following elements: an enhanced cauliflower mosaic virus 35S promoter and leader sequence (EXP-CaMV. 35S-enh:1:2, SEQ ID NO: 55) operably linked to 5' of a synthetic coding sequence encoding β -glucuronidase (GOI-ec. UidA+St. LS1:3, SEQ ID NO: 50) containing a processable intron derived from the potato light-induced tissue specific ST-LS1 gene (GenBank accession number: X04753), operably linked to 5' of a 3' termination region (T-AGRtu. Nos:13, SEQ ID NO: 48).

EXP-Gm.Rsp-1:1 driven efficient automatic excision in stably transformed soybean plants.

Soybean plants were transformed with the binary construct as described above, wherein EXP (EXP-gm. Rsp-1:1 (SEQ ID NO: 20)) driven the expression of the Cre-recombinase transgene. The Cre-recombinase expression cassette consists of EXP (EXP-Gm. Rsp-1:1 (SEQ ID NO: 20)) consisting of the following elements: a promoter (P-Gm.Rsp-1-1:1:1,SEQ ID NO:21) operably linked to 5 'of a leader sequence (L-Gm.Rsp-1-1:1:1,SEQ ID NO:22) operably linked to 5' of a synthetic coding sequence encoding Cre-recombinase operably linked to 5 'of a 3' termination region (T-At.Cdc45:1, SEQ ID NO: 12).

The binary plant transformation constructs described above are used to transform soybean plant cells by agrobacterium-mediated transformation, as is well known in the art. The resulting transformed plant cells were induced under selection for spectinomycin to form whole soybean plants.

Fifty R are allowed ₀ Single copy events self-pollinate. And then useAnalysis of the obtained R by assay ₁ The presence of Cre, aadA and GUS transgenes in plants. Also use +.> The assay determines the zygosity of the GUS transgene cassette. Will be for fifty R ₀ GUS positive (GUS+) and unlabeled (aadA-) R for each of the events ₁ The percentages of the offspring are presented in table 4 below.

TABLE 4 GUS positive (GUS+) and unlabeled (aadA-) R in stably transformed soybean plants with EXP-Gm.Rsp-1:1 driven automatic excision ₁ Percentage of offspring

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As can be seen from Table 4 above, except for one R ₀ Except for event (event-26), all events produced a marker-free R after self-pollination ₁ And (5) offspring. Some R ₀ Events provide a large proportion of unlabeled R ₁ Progeny plants, many of which are homozygous for the GUS marker expression cassette, such as event-12, event-15, event-18, event-20, event-37, event-41, event-44, event-46 and event-48. The P-Gm.Rsp-1:1:1 promoter (SEQ ID NO: 21) contained in EXP-Gm.Rsp-1:1 (SEQ ID NO: 20) provides efficient automatic excision in stably transformed soybean plants.

The germ line preferred Dmc1 promoter does not drive efficient automatic excision in stably transformed soybean plants.

The Dmc1 promoter, which has been demonstrated to be preferred by Arabidopsis germline, drives efficient automatic excision in transformed Arabidopsis plants. To see if such a promoter is capable of driving automatic excision in stably transformed soybean plants, binary plant transformation vectors as described above were constructed in which EXP (EXP-at. Dmc1+Zm. DnaK:1:1 (SEQ ID NO: 40)) was operably linked to 5' of the synthetic coding sequence encoding Cre-recombinase.

Allow for selection of R ₀ Single copy events self-pollinate. And then useAnalysis of the obtained R by assay ₁ The presence of Cre, aadA and GUS transgenes in plants. Also use +.>The assay determines the zygosity of the GUS transgene cassette. Will be directed to the R of the selection ₀ GUS positive (GUS+) and unlabeled (aadA-) R for each of the events ₁ The percentages of the offspring are presented in table 5 below.

TABLE 5 GUS positive (GUS+) and unlabeled (aadA-) R in stably transformed soybean plants with automatic excision driven by the Dmc1 promoter ₁ Percentage of offspring.

As can be seen from Table 5 above, there are only a few R ₀ Single copy events provide unmarked R ₁ And (5) offspring. Twenty R ₀ Event(s)Only three events in (a) produced homozygous GUS positive, unlabeled plants. Compared to EXP-Gm.Rsp-1:1 (SEQ ID NO: 20), R comprising EXP-at.Dmc1+Zm.DnaK 1:1 (SEQ ID NO: 40) driving Cre-recombinase ₀ The event did not provide efficient automatic excision in stably transformed plants.

The introduction of the Kozak consensus sequence into 3' of EXP-gm. Rsp-1:1 did not significantly affect the efficiency of automatic excision in stably transformed soybean plants.

The leader sequence of EXP-Gm.Rsp-1:1 (L-Gm.Rsp-1-1:1:1,SEQ ID NO:22) comprises a ninety three base pair small fragment of the Open Reading Frame (ORF) of protein 9 containing the soybean BURP domain. The small ORF fragment is in frame with the Cre-recombinase coding sequence, so this fragment does not interfere with R ₁ Efficient automatic excision in progeny plants. To shift the bias of translation initiation to the initiation codon of the Cre-recombinase coding sequence, a small Kozak consensus sequence (5 '-GCAAAA-3) was operably linked to 3' of EXP-Gm.Rsp-1:1 (EXP-Gm.Rsp-1:1+Kozak) according to Nakagawa et al, 2007 (Nakagawa et al, (2007) Di versity of preferred nucleotide sequences around the translation initiati on codon in eukaryote genome.nucleic Acids Research,36 (3): 861-871).

Four binary plant transformation constructs were similarly constructed as described above, each comprising EXP-Gm.Rsp-1:1 (SEQ ID NO: 20) operably linked to 5' of the synthetic coding sequence encoding Cre-recombinase. One construct also comprises a 3' small Kozak consensus sequence operably linked to EXP-gm.rsp-1:1 (EX P-gm.rsp-1:1+kozak). The other two constructs contained truncated variants of T-At.Cdc45:1 (SEQ ID NO: 12), T-At.Cdc45:3 (SEQ ID NO: 23) and T-At.Cdc45:4 (SEQ ID NO: 24). GUS expression cassettes outside the LoxP site are replaced with expression cassettes expressing herbicide tolerance genes.

Allow for selection of R ₀ Single copy events self-pollinate. Then makeBy usingAnalysis of the obtained R by assay ₁ The presence of Cre, aadA and herbicide tolerance transgenes in plants. Also use +.>The assay determines the zygosity of Herbicide Tolerance (HT) transgene cassettes. Will be directed to the R of the selection ₀ Homozygous HT positive and marker-free R for each of the events ₁ The percentages of the offspring are presented in table 6 below.

TABLE 6 homozygous HT positive and marker-free R in stably transformed soybean plants ₁ Percentage of offspring.

As can be seen in Table 6 above, the introduction of the Kozak consensus sequence into the 3' of EXP-Gm.Rsp-1:1 had minimal effect on the efficiency of automatic excision in stably transformed soybean plants (23%, 29% relative to no Kozak consensus sequence). Truncated variants of T-at.Cdc45:1' UTR reduce the efficiency of automatic excision. However, for both truncated variants of T-At.Cdc45:1, automatic excision still occurs.

Introduction of introns 3' of EXP-gm. Rsp-1:1 significantly reduced automatic excision in stably transformed soybean plants.

Introns are known in the art to improve expression of transgenes. To evaluate the role of introns in automatic excision, the intron I-at. AtpE 1 (SEQ ID NO: 26) was cloned into 3' of EXP-Gm. R sp-1:1 in binary plant transformation constructs similar to those described above. In addition to Cre, aadA and GUS transgene cassettes, a third transgene cassette was cloned between LoxP sites for expression of sucrose phosphorylase genes; visual marking. When splA expression cassettes are present in the seed, the seed will shrink. Such visual markers allow for a rapid assessment of the presence of marker gene cassettes that were not removed by automated excision. The expression cassette consists of the following elements: seed enhanced EXP (EXP)Vf. Usp88-enh:1:1 (SEQ ID NO: 60)), said EXP being operably linked to 5' of a coding sequence encoding a sucrose phosphorylase (CR-AGRtu. SplA-C58:1:3, SEQ ID NO: 61), said coding sequence being operably linked to 5' of a 3' termination region (T-AGRtu. Nos. 13, SEQ ID NO: 48). Allow for selection of R ₀ Single copy events self-pollinate. UsingThe assay determines the presence of and zygosity of the GUS transgene cassette. By visual inspection R ₁ Offspring seeds and observed for lack of the shrunken seed phenotype, the lack of Cre, aadA and splA expression cassettes was determined. It was inferred that those seeds with the appearance of shrunken seeds still contained Cre, aadA and splA expression cassettes. Will be directed to the R of the selection ₀ GUS positive (GUS+) and unlabeled (aadA-) R for each of the events ₁ The percentages of the offspring are presented in table 7 below.

TABLE 7 GUS positive (GUS+) and unlabeled (aadA-) R in stably transformed soybean plants with EXP-Gm.Rsp-1:1 and I-AtpE:1 driven automatic excision ₁ Percentage of offspring.

As can be seen in Table 7 above, the addition of intron I-at. AtpE 1 (SEQ ID NO: 26) to 3' of EXP-Gm. Rsp-1:1 significantly reduced the efficiency of automatic excision in stably transformed soybean plants compared to the results without introns (see Table 4 above). Minority R ₀ The event produced a homozygous GUS positive unlabeled event. In contrast, as seen in example 5 below, the introduction of the I-At.AtpE 1 intron to 3' of EXP-Gm.Rsp-1:1 enhances the ability of EXP-Gm.Rsp-1:1 to effectively drive automatic excision in cotton plants.

Example 5

Automatic excision in stably transformed cotton plants is driven when the soybean Rsp-1 promoter is operably linked to the intron I-At.AtpE:1

Cotton plants were transformed with the same binary plant transformation construct comprising intron I-at. AtpE 1 (SEQ ID NO: 26) cloned into the 3' of EXP-Gm. Rsp-1:1 as described in the previous examples. Allow for selection of R ₀ Single copy events self-pollinate. Using The assay determines the presence of and zygosity of the GUS transgene cassette. By visual inspection R ₁ Offspring seeds and observed for lack of the shrunken seed phenotype, the lack of Cre, aadA and splA expression cassettes was determined. Will be directed to the R of the selection ₀ GUS positive (GUS+) and unlabeled (aadA-) R for each of the events ₁ The percentages of the offspring are presented in table 8 below.

TABLE 8 GUS positive (GUS+) and unlabeled (aadA-) R in stably transformed cotton plants with EXP-Gm.Rsp-1:1 and I-AtpE:1 driven automatic excision ₁ Percentage of offspring.

As can be seen from Table 8 above, cloning the I-at. AtpE 1 (SEQ ID NO: 26) intron to 3' of EXP-Gm. Rsp-1:1 resulted in efficient automatic excision in stably transformed cotton plants, compared to the results obtained using the same binary plant transformation construct in soybean. All but one event (event-3) provides an unlabeled R ₁ Offspring seed and homozygous GUS positive, unlabeled negative R ₁ Offspring seed.

Example 6

Arabidopsis CDC45 promoter drives auto excision in stably transformed cotton plants

Cotton plants were transformed with constructs, particularly plant binary transformation constructs comprising EXP (EXP-at. Cdc45:1:1 (SEQ ID NO: 9)) presented in example 3, driving Cre-recombinase expression, and used to assess the ability and efficiency of automatic excision of Cre-recombinase expression cassettes and marker gene expression cassettes.

Binary plant transformation constructs as described in example 4 were cloned using methods well known in the art. EXP (EXP-At.Cdc45:1:1 (SEQ ID NO: 9)) is operably linked to 5' of a synthetic coding sequence encoding Cre-recombinase (GOI-P1. Cre-St.LS1:1:1, SEQ ID NO: 43) operably linked to 5' of a 3' termination region (T-At.Cdc45:1, SEQ ID NO: 12).

Cotton plant cells were transformed with the binary plant transformation constructs described above by agrobacterium-mediated transformation. Under the selection of spectinomycin, the resulting transformed plant cells are induced to form whole cotton plants.

Allow for selection of R ₀ Single copy events self-pollinate. And then useAnalysis of the obtained R by assay ₁ The presence of Cre, aadA and GUS transgenes in plants. Also use +.>The assay determines the zygosity of the GUS transgene cassette. Will be directed to the R of the selection ₀ GUS positive (GUS+) and unlabeled (aadA-) R for each of the events ₁ The percentages of the offspring are presented in table 9 below.

TABLE 9 GUS positive (GUS+) and unlabeled (aadA-) R in stably transformed cotton plants with automatic excision driven by the Arabidopsis CDC45 promoter ₁ Percentage of offspring.

As can be seen from Table 9 above, except for one R ₀ Except for event (event-14), all events produced GUS positive (GUS+) and unlabeled (aadA-) R ₁ And (5) offspring. Some R ₀ Events produce a significant proportion of unlabeled and homozygous unlabeled events, such as event-2, event-7, and event-11. The CDC45 promoter (P-at. Cdc45-1:1:1,SEQ ID NO:10) contained in EXP-at. Cdc45:1:1 (SEQ ID NO: 9) was able to efficiently drive automatic excision in stably transformed cotton plants.

Example 7

Chimeric P-vf. Usp 88-chimeric promoters drive efficient automatic excision in stably transformed canola plants

The canola plants were transformed with constructs, particularly plant binary transformation constructs comprising EXP (EXP-vf. Usp88-enh:1:1 (SEQ ID NO: 60)) to drive Cre-recombinase expression, and used to assess the ability and efficiency of automatic excision of Cre-recombinase expression cassettes and marker gene expression cassettes. EXP (EXP-vf. Usp88-enh:1:1 (SEQ ID NO: 60)) consists of the following elements: chimeric promoter P-vf. Usp 88-chimeric (SEQ ID NO: 61) operably linked to 5' of leader sequence L-vf. Usp-1:1:1 (SEQ ID NO: 62).

Transforming a canola plant with a binary plant transformation construct comprising the following three transgene expression cassettes: cre-recombinase expression cassette and a selectable marker expression cassette (aadA) for selecting transformed soybean cells flanked by two loxP sites (RS-P1. Lox1:1, SEQ ID NO: 44); and a third expression cassette outside the LoxP site for expressing a beta-Glucuronidase (GUS) transgene. The Cre-recombinase expression cassette consists of the following elements: EXP (EXP-vf. Usp88-enh:1:1 (SEQ ID NO: 60)), operably linked to 5' of a synthetic coding sequence encoding Cre-recombinase (GOI-P1. Cre-St. LS1:1:1, SEQ ID NO: 43) containing a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (GenBank accession number X04753), operably linked to 5' of a 3' termination region (T-Br. Snap2-1:3:6,SEQ ID NO:59). The transformation selection marker cassette aadA consists of the following elements: EXP (EXP-at. Act7:2, SEQ ID NO: 53) operably linked to 5' encoding a chloroplast-targeted Tn7 adenylyl transferase coding sequence (GOI-at. ShkG-CTP2+ec. AadA-SPC/STR:1:1, SEQ ID NO: 54) that confers resistance to spectinomycin and is used to select transformed plant cells, said coding sequence being operably linked to 5' of a 3' termination region (T-AGRt u. Nos. 13, SEQ ID NO: 48). The GUS transgene expression cassette consists of the following elements: an enhanced cauliflower mosaic virus 35S promoter and leader sequence (EXP-CaMV. 35S-enh:1:2, SEQ ID NO: 55) operably linked to 5' of a synthetic coding sequence encoding β -glucuronidase (GOI-ec. UidA+St. LS1:3, SEQ ID NO: 50) containing a processable intron derived from the potato light-induced tissue specific ST-LS1 gene (GenBank accession number: X04753), operably linked to 5' of a 3' termination region (T-AG Rtu. Nos:13, SEQ ID NO: 48).

The binary plant transformation constructs described above are used to transform canola plant cells by agrobacterium-mediated transformation, as is well known in the art. The resulting transformed plant cells were induced to form whole canola plants under the selection of spectinomycin (aadA).

Allow for selection of R ₀ Single copy events self-pollinate. And then useAnalysis of the obtained R by assay ₁ Presence of aadA and GUS transgenes in seeds. Also using GUS transgene +.>Determination of R by assay ₁ Zygosity of seeds for the integration construct. According to each selected R ₀ Event selfing, eighty-eight R was determined ₁ Seed. Table 10 below shows the number of GUS+/aadA-events of hemizygous and homozygous observed for each event seed sample.

Table 10. Number and percentage of GUS+/aadA-R1 progeny seeds of hemizygous and homozygous samples in stably transformed canola plants with automatic excision driven by chimeric P-vf. Usp 88-chimeric promoter.

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As can be seen from Table 9 above, except for one R ₀ Except for event (event-3), all events produced homozygous GUS positive (GUS+) and unlabeled (aadA-) R ₁ And (5) offspring. Event-3 may be a chimeric event in which some cells are not transformed, as happens occasionally in plant transformation. Some R ₀ Events produce a significant proportion of unmarked events of hemizygous events, such as event-2, event-4, event-5, event-7, and event-8. EXP (EXP-vf. Usp88-enh:1:1 (SEQ ID NO: 61)) comprising the chimeric promoter P-vf. Usp 88-chimera (SEQ ID NO: 61) was able to efficiently drive automatic excision in stably transformed canola plants.

Example 8

Automatic excision of EXP-Gm.Rsp-1:1 driven selection markers and genome editing transgene cassettes in stably transformed soybean plants

Soybean plants were transformed with constructs, particularly plant binary transformation constructs comprising EXP (EXP-Gm. Rsp-1:1 (SEQ ID NO: 20)) driving Cre-recombinase expression, and used to evaluate the ability and efficiency of automatic excision of Cre-recombinase expression cassettes, marker gene expression cassettes, and expression cassettes for soybean genome editing.

Transforming a soybean plant with a binary plant transformation construct comprising the following five transgenic expression cassettes; cre-recombinase expression cassette; a selectable marker expression cassette (aadA) for selecting transformed soybean cells; an expression cassette for expressing a Cpf1 CRISPR-associated nuclease; an expression cassette for expression of guide RN A flanked by two LoxP sites (RS-P1. Lox1:1, SEQ ID NO: 44); and a fifth expression cassette located outside the LoxP site for expressing genes of agronomic interest. The Cre-recombinase expression cassette consists of the following elements: EXP (EXP-Gm. Rsp-1:1 (SEQ ID NO: 20)) operably linked to 5 'of a synthetic coding sequence encoding Cre-recombinase (GOI-P1. Cre-St. LS1:1:1, SEQ ID NO: 43) containing a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (GenBank accession number X04753) operably linked to a 3' termination region (T-at. Cdc45:3, SE Q ID NO: 23). The transformation selection marker cassette aadA consists of the following elements: EXP (EXP-at. Act7:2, SEQ ID NO: 54) operably linked to 5' encoding a chloroplast-targeted Tn7 adenylyl transferase coding sequence (GOI-at. ShkG-CTP2+ec. AadA-SPC/STR:1:1, SEQ ID NO: 54) that confers resistance to spectinomycin and is used to select transformed plant cells, said coding sequence being operably linked to 5' of a 3' termination region (T-AGRtu. Nos. 13, SEQ ID NO: 48). The Cpf1 expression cassette consists of the following elements: constitutively expressed EXP is provided operably linked to the 5' of a nuclear targeted Cpf1 coding sequence operably linked to the 5' of a 3' plant termination region. The guide RNA expression cassette consists of the following elements: an RNA polymerase III promoter operably linked to 5' of a guide RNA coding sequence operably linked to 5' of a 3' RNA polymerase III termination region. All four expression cassettes are flanked by LoxP sites. The fifth transgene expression cassette consists of the following elements: plants are constitutively EXP operably linked to 5' of a coding sequence of a gene encoding an agronomic benefit operably linked to 5' of a 3' plant termination region.

The guide RNA transgene cassette comprises a guide RNA designed to provide site-specific integration of the construct at a specific location within the soybean genome. The construct is designed such that upon integration of the construct into R ₁ The specific region of the soybean genome of the generation soybean seed is then provided with Cre, aadA, cpf1 and automatic excision of the guide RNA transgene expression cassette. After automatic excision, at R ₁ Only the fifth transgenic expression cassette for expression of genes of agronomic interest should be maintained in the transgenic soybean plants.

All R's are determined ₀ Transgenic copy number of transgenic event. They were also analyzed for site-specific integration using flanking PCR assays, in which amplification was performed across the target site ligation. By bringing selected R ₀ Transgenic events selfed to generate R ₁ Seed generation.Determination of R ₁ Random samples of seed generation were marked and genome editing cassettes were present. Table 11 below shows the number and percentage of seeds without hemizygous and homozygous markers and genome editing cassettes from eight selected events.

Table 11 number and percentage of R1 progeny seeds without hemizygous and homozygous markers and genome editing cassettes sampled from stably transformed soybean plants with EXP-Gm.Rsp-1:1 driven automatic excision.

As can be seen in Table 11 above, EXP (EXP-Gm. Rsp-1:1 (SEQ ID NO: 20)) is capable of driving the automatic excision of markers and genome editing cassettes with an efficiency similar to that shown in example 4 above.

Example 9

EXP-Gm.Nmh7:1 driven automatic excision of selectable markers and genome editing transgene cassettes in stably transformed soybean plants

Soybean plants were transformed with constructs, particularly plant binary transformation constructs comprising EXP (EXP-Gm. Nmh7:1 (SEQ ID NO: 64)) driving Cre-recombinase expression, and used to evaluate the ability and efficiency of automatic excision of Cre-recombinase expression cassettes, marker gene expression cassettes, and expression cassettes for soybean genome editing.

Transforming a soybean plant with a binary plant transformation construct comprising five transgenic expression cassettes; cre-recombinase expression cassette; a selectable marker expression cassette (aadA) for selecting transformed soybean cells; an expression cassette for expressing a Cpf1 CRISPR-associated nuclease; an expression cassette for expressing a guide RNA flanked by two LoxP sites (RS-P1. Lox1:1, SEQ ID NO: 44); and a fifth expression cassette located outside the LoxP site for expressing genes of agronomic interest. All expression cassettes were similar to those described in example 7 above. The Cre-recombinase expression cassette consists of the following elements: EXP (EXP-Gm. Nmh7:1 (SEQ ID NO: 64)) operably linked to 5' of a synthetic coding sequence encoding Cre-recombinase (GOI-P1. Cre-St. LS1:1:1, SEQ ID NO: 43) containing a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (GenBank accession number X04753) operably linked to 5' of a 3' termination region (T-Gb. E6-3b:1:1,SEQ ID NO:67). Two different constructs were used in the transformation. Each construct comprises a different constitutive promoter driving expression of the Cpf1 CRISPR-associated nuclease.

All R's are determined ₀ Transgenic copy number of transgenic event. They were also analyzed for site-specific integration using flanking PCR assays, in which amplification was performed across the target site ligation. By bringing selected R ₀ Transgenic events selfed to generate R ₁ Seed generation. Determination of R ₁ Random samples of seed generation were marked and genome editing cassettes were present. Table 12 below shows the number and percentage of seeds without hemizygous and homozygous markers and genome editing cassettes from eight selected events.

Table 12 number and percentage of R1 progeny seeds without hemizygous and homozygous markers and genome editing cassettes sampled from stably transformed soybean plants with EXP-Gm.Nmh7:1 driven automatic excision.

As can be seen in Table 12 above, two events derived from transformation using each construct resulted in R without hemizygous markers and genome editing cassettes ₁ And (5) offspring. Event 2, resulting from transformation using construct-1, even produced several R's without homozygous markers and genome editing cassettes ₁ And (5) offspring.

Example 10

Automatic excision of selectable markers and genome editing transgene cassettes in stably transformed maize plants driven by maize and rice CDC45-1 promoters

Maize plants are transformed with a construct, in particular a plant binary transformation construct comprising EXP-Zm.Cdc45-1+Zm.DnaK:1:1 (SEQ ID NO: 1) or EXP-Os.Cdc45-1:1:1 (SEQ ID NO: 4) driving Cre-recombinase expression.

A maize plant is transformed with a construct comprising at least five expression cassettes. The construct comprises a Cre-recombinase expression cassette, at least one selectable marker cassette, an expression cassette for expressing a CRISP R-associated nuclease such as Cpf1, at least one guide RNA cassette; all these cassettes flank the loxp site. The construct further comprises at least one expression cassette outside the LoxP site for expression of genes of agronomic interest. Each construct contained a Cre-recombinase expression cassette similar to that described in example 2, with EXP-Zm.Cdc45-1+Zm.DnaK:1:1 (SEQ ID NO: 1) or EXP-Os.Cdc45-1:1:1 (SEQ ID NO: 4) used to drive Cre-recombinase expression.

Transformation of maize plants with either of the two constructs and selection of R using molecular assays ₀ Transgenic events to determine copy number, insert integrity. If the construct is designed to provide site-specific integration of the construct; confirmation of the insertion site within the maize genome is confirmed by amplification of the insertion site ligation. By bringing selected R ₀ Transgenic event selfing to generate R ₁ Seed generation. Seeds or germinated offspring were analyzed for removal of Cre, marker, cpf1 and guide RNA cassette. They were also analyzed for the presence of one or more expression cassettes for one or more agronomically beneficial genes.

Having illustrated and described the principles of the present invention, it will be apparent to those skilled in the art that the invention may be modified in arrangement and detail without departing from such principles. We claim all such modifications as fall within the spirit and scope of the following claims. All publications and published patent documents cited herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A recombinant DNA construct comprising a DNA regulatory sequence comprising:

a. a sequence having at least 80% sequence identity to any one of SEQ ID NOs 1-26, 59-62 and 64-66;

b. a sequence comprising any one of SEQ ID NOs 1-26, 59-62 and 64-66; and

c. (i) A fragment of any one of SEQ ID NOs 1-26, 59-62 and 64-66 or (ii) any sequence having at least 80% sequence identity to any one of SEQ ID NOs 1-26, 59-62 and 64-66, wherein said fragment has gene regulatory activity;

Wherein the DNA regulatory sequence is operably linked to a heterologous transcribable DNA sequence encoding a site-specific recombinase.

2. The recombinant DNA molecule of claim 1, wherein said DNA regulatory sequence has at least 90% sequence identity to the DNA sequence of any one of SEQ ID NOs 1-26, 59-62 and 64-66.

3. The recombinant DNA molecule of claim 1 or 2, wherein said DNA regulatory sequence has at least 95% sequence identity to the DNA sequence of any one of SEQ ID NOs 1-26, 59-62 and 64-66.

4. The recombinant DNA molecule of claim 1, 2 or 3, wherein said DNA regulatory sequence has gene regulatory activity.

5. The recombinant DNA molecule of any one of claims 1-4, wherein the DNA regulatory sequence is a germline-preferred promoter.

6. The recombinant DNA molecule of any one of claims 1-5, wherein the germline-preferred promoter is selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 10, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 17, SEQ ID NO. 21 and SEQ ID NO. 65, or a sequence having at least 80% sequence identity to any of SEQ ID NO. 2, 5, 7, 10, 13, 14, 17, 21 and 65.

7. The recombinant DNA molecule of any one of claims 1-6, wherein the germline-preferred promoter is a CDC45 promoter.

8. The CDC45 promoter of any one of claims 1-7, wherein the CDC45 promoter is selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 5 and SEQ ID NO. 10, or a sequence having at least 80% sequence identity to any of SEQ ID NO. 2, 5 and 10.

9. The recombinant DNA molecule of any one of claims 1-5, wherein the DNA regulatory sequence is SEQ ID No. 60 or a sequence having at least 80% sequence identity to SEQ ID No. 60.

10. The recombinant DNA molecule of any one of claims 1-9, wherein the site-specific recombinase is selected from the group consisting of: cre-recombinase, flp-recombinase, R-recombinase and Gin-recombinase.

11. The recombinant DNA molecule of any one of claims 1-10, wherein the site-specific recombinase is Cre-recombinase.

12. The recombinant DNA construct of any one of claims 1-11, further comprising one or both of the following expression cassettes: a selectable marker transgene; and/or transgenes of agronomic interest.

13. The recombinant DNA construct according to any one of claims 1-12, further comprising a pair of site-specific recombination site sequences flanking one or both transcribable DNA sequences encoding the site-specific recombinase and/or the selectable marker transgene, wherein the site-specific recombination site is cleavable by the site-specific recombinase.

14. The recombinant DNA construct according to claim 13, wherein said pair of site-specific recombination site sequences are oriented in a head-to-tail arrangement.

15. The recombinant DNA construct according to claim 13 or 14, wherein said selectable marker transgene confers resistance to a herbicide or antibiotic.

16. The recombinant DNA construct according to any one of claims 13, 14 or 15, wherein said pair of site-specific recombination site sequences are each selected from the group consisting of: loxP, lox.TATA-R9, FRT, RS and GIX.

17. The recombinant DNA construct according to any one of claims 12-16, wherein the pair of site-specific recombination sites are each LoxP or lox.tata-R9 sites.

18. The recombinant DNA construct according to any one of claims 12-17, wherein said pair of site-specific recombination site sequences each comprises SEQ ID No. 44 or SEQ ID No. 45.

19. The recombinant DNA construct according to any one of claims 12-18, wherein said agronomically beneficial transgene confers herbicide tolerance in a plant.

20. The recombinant DNA construct according to any one of claims 12-18, wherein said agronomically beneficial transgene confers insect resistance or disease resistance in a plant.

21. The recombinant DNA construct according to any one of claims 12-18, wherein said agronomically beneficial transgene confers increased yield or stress tolerance in a plant.

22. The recombinant DNA construct according to any one of claims 12-21, wherein said agronomically beneficial transgene encodes a dsRNA, miRNA or siRNA.

23. The recombinant DNA construct of any one of claims 1-22, further comprising one or both of: an expression cassette encoding a guide RNA; and/or an expression cassette encoding a site-specific nuclease.

24. The recombinant DNA construct according to claim 23, further comprising a pair of site-specific recombination site sequences flanking one or more transcribable DNA sequences encoding the site-specific recombinase, a selectable marker transgene, an expression cassette encoding the guide RNA, and/or an expression cassette encoding the site-specific nuclease, wherein the site-specific recombination site is cleavable by the site-specific recombinase.

25. The recombinant DNA construct according to claim 23 or 24, wherein said guide RNA comprises a targeting sequence that targets a sequence in the genome of a eukaryotic cell for genome editing or site-specific integration.

26. The recombinant DNA construct according to claim 25, wherein said eukaryotic cell is a plant cell.

27. The recombinant DNA construct according to any one of claims 23-26, comprising two or more expression cassettes encoding two or more guide RNAs.

28. The recombinant DNA construct according to any one of claims 23-27, wherein there are two, three, four, five, six, seven, eight, nine or ten different expression cassettes encoding guide RNAs.

29. The recombinant DNA construct according to any one of claims 23-28, wherein said site-specific nuclease is an RNA-guided endonuclease.

30. The recombinant DNA construct according to any one of claims 23-29, wherein said RNA-guided endonuclease is selected from the group consisting of: cas1, cas1B, cas2, cas3, cas4, cas5, cas6, cas7, cas8, cas9, cas10, cpf1, cys2, csy3, cse1, cse2, csc1, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmr1, cmr3, cmr4, cmr5, cmr6, csb1, csb2, csb3, csx17, csx14, csx10, csx16, csaX, csx3, csx1, csx15, csf1, csf2, csf3, csf4, casX and CasY.

31. The recombinant DNA construct of claim 30, wherein the RNA-guided endonuclease is Cas9 or Cpf1.

32. A DNA molecule or vector comprising the recombinant DNA construct of any one of claims 1-31.

33. A DNA transformation vector comprising the recombinant DNA construct of any one of claims 1-31 and a T-DNA segment bounded by a left border and a right border.

34. The DNA transformation vector of claim 33, wherein the transcribable DNA sequence encoding the site-specific recombinase is located between the left and right boundaries of the T-DNA segment.

35. A DNA transformation vector comprising the recombinant DNA construct of any one of claims 12-31 and a T-DNA segment having a left border and a right border, wherein one or more of the transcribable DNA sequence encoding the site-specific recombinase, the selectable marker transgene, and/or the agronomically beneficial transgene is located between the left border and the right border of the T-DNA segment.

36. A DNA transformation vector comprising the recombinant DNA construct of any one of claims 23-31 and a T-DNA segment having a left border and a right border, wherein one or more of the transcribable DNA sequence encoding the site-specific recombinase, the selectable marker transgene, the agronomically beneficial transgene, the expression cassette encoding the guide RNA, and/or the expression cassette encoding the site-specific nuclease is located between the left border and the right border of the T-DNA segment.

37. A transgenic plant, plant part or plant cell comprising the recombinant DNA construct of any one of claims 1-31.

38. The transgenic plant, plant part, or plant cell of claim 37, wherein the recombinant DNA construct is stably transformed into the genome of the transgenic plant, plant part, or plant cell.

39. The transgenic plant, plant part or plant cell according to claim 37 or 38, wherein the transgenic plant, plant part or plant cell is a maize, soybean, cotton or canola plant, plant part or plant cell.

40. A bacterial cell comprising the recombinant DNA construct of any one of claims 1-31, the DNA molecule or vector of claim 32, or the transformation vector of any one of claims 33-36.

41. A method of producing a transgenic plant or plant part comprising:

a. transforming plant cells of an explant with a DNA molecule or vector comprising the recombinant DNA construct of any one of claims 1-30 to produce one or more transformed plant cells comprising the recombinant DNA construct stably transformed into the genome of the one or more transformed plant cells;

b. Regenerating or developing a transgenic plant from said explant, wherein said transgenic plant comprises said recombinant DNA construct stably transformed into the genome of one or more cells of said transgenic plant.

42. The method of claim 41, wherein the plant cell is transformed via Agrobacterium-mediated transformation or rhizobium-mediated transformation.

43. The method of claim 41, wherein the plant cell is transformed via particle-mediated transformation or particle bombardment-mediated transformation.

44. The method of claim 41, wherein the transgenic plant and plant cell are maize, soybean, cotton or canola plants and plant cells, respectively.

45. The method of claim 41, further comprising:

a. isolating or harvesting a plant part from said transgenic plant.

46. A method for excision of an expression cassette from the genome of a transgenic plant comprising:

a. transforming a plant cell with a DNA molecule or vector comprising the recombinant DNA construct of any one of claims 13-29 to produce one or more transformed plant cells comprising the recombinant DNA construct stably transformed into the genome of the one or more transformed plant cells;

b. Regenerating or developing a transgenic plant at least partially from the one or more stably transformed plant cells;

c. crossing the transgenic plant with itself or another plant; and

d. selecting one or more progeny plants, wherein one or both of the transcribable DNA sequence encoding the site-specific recombinase and/or a selectable marker transgene between a pair of site-specific recombination site sequences of the recombinant DNA construct is excised and no longer present in the genome of the progeny plant.

47. The method according to claim 46,

wherein the recombinant DNA construct further comprises one or both of the following expression cassettes located between the pair of DNA site-specific recombination site sequences of the recombinant DNA construct: an expression cassette encoding a guide RNA and/or an expression cassette encoding a site-specific nuclease, and wherein one or more progeny plants are selected, wherein one or more of the transcribable DNA sequence encoding the site-specific recombinase, the selectable marker transgene, the expression cassette encoding the guide RNA, and/or the expression cassette encoding the site-specific nuclease of the recombinant DNA construct is excised and no longer present in the genome of the progeny plants.

48. The method of claim 46, wherein the transgenic plant and plant cell are maize, soybean, cotton or canola plants and plant cells, respectively.

49. The method of claim 46, further comprising:

a. plant parts are isolated or harvested from one or more of the progeny plants.

50. The method of claim 46, further comprising:

a. crossing one or more of said progeny plants with itself or another plant.